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

Composition and method for generating recombinant baculovirus in insect cell, and use Download PDF

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WO2023221240A1
WO2023221240A1 PCT/CN2022/101538 CN2022101538W WO2023221240A1 WO 2023221240 A1 WO2023221240 A1 WO 2023221240A1 CN 2022101538 W CN2022101538 W CN 2022101538W WO 2023221240 A1 WO2023221240 A1 WO 2023221240A1
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baculovirus
recombinant
expression cassette
gene expression
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Chinese (zh)
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肖何
何晓斌
黄刚
胡颖
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睿征医药科技(武汉)有限公司
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Definitions

  • 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.
  • 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.
  • MNPV Autographa californica multinuclear polyhedrosis virus
  • BEVS Baculovirus expression system
  • BEVS Baculovirus expression system
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the composition is capable of homologous recombination in insect cells. to produce recombinant baculovirus.
  • the first functional fragment is the cap gene expression cassette of AAV and the rep gene expression cassette of AAV.
  • 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'.
  • 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'.
  • 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.
  • the packaging-deficient baculovirus plasmid is a recombinant bacmid containing an AAV ITR core expression element with a foreign gene.
  • the recombinant bacmid is obtained by Tn7 transposition mediated by a baculovirus transfer vector.
  • the first functional fragment is an AAV ITR core expression element carrying an exogenous gene.
  • the packaging-deficient baculovirus plasmid is a recombinant bacmid containing an AAV cap gene expression cassette and an AAV rep gene expression cassette.
  • the recombinant bacmid is obtained by Tn7 transposition mediated by a baculovirus transfer vector.
  • the first functional fragment is a reporter gene.
  • the first functional fragment is a nucleotide sequence encoding a therapeutic gene product.
  • 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
  • the second complementation sequence It is the sequence different from the first complement sequence in the CNE sequence and the NAE sequence.
  • the first functional fragment is the cap gene expression cassette of AAV and the rep gene expression cassette of AAV
  • the second insertion sequence also includes a second functional fragment
  • the second functional fragment is a gene containing an exogenous gene.
  • AAV ITR core expression element is the first functional fragment.
  • compositions for preparing recombinant baculovirus and/or recombinant adeno-associated virus in insect cells are provided.
  • an insect cell comprising any of the above compositions.
  • a method for growing or producing recombinant baculovirus in vitro comprising co-transfecting insect cells with any of the above compositions and culturing the insect cells.
  • a method for growing or producing recombinant adeno-associated viruses in vitro comprising co-transfecting insect cells with any of the above compositions and culturing the insect cells.
  • 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.
  • 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 ⁇ CNE-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 ⁇ CNE-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 ⁇ NAE-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 ⁇ NAE-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 ⁇ CNE-Bac and the recombinant DNA fragments Ac83-CNE-GFP, Ac126-CNE-GFP and Ac152-CNE-GFP in Example 4 of the present invention.
  • Figure 14 shows the results obtained after co-transfecting insect host cells with the defective baculovirus vector ⁇ CNE-Bac and the recombinant DNA fragments Ac83-CNE-GFP, Ac126-CNE-GFP and Ac152-CNE-GFP in Example 4 of the present invention.
  • Figure 15 shows the results produced by co-transfecting insect host cells with the defective baculovirus vector ⁇ NAE-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 ⁇ NAE-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 ⁇ CNE-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. .
  • 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.
  • plural means at least two, such as two, three, etc., unless otherwise expressly and specifically limited.
  • severeal means at least one, such as one, two, etc., unless otherwise expressly and specifically limited.
  • 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. .
  • 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.
  • 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.
  • CNE non-protein-coding element
  • 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 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.
  • 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.
  • the first complement sequence is the CNE sequence
  • the packaging-defective baculovirus plasmid lacks the NAE sequence in the baculovirus genome
  • the first complement sequence is the NAE sequence
  • 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.
  • 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.
  • 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.
  • first homology arm and the second homology arm 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.
  • 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.
  • the rescue recombinant DNA provided by the present invention has no restriction on the insertion site.
  • 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.
  • 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, ⁇ -galactosidase, ⁇ -glucuronidase, Renilla luciferase, firefly luciferase or alkaline phosphatase.
  • fluorescent protein green Fluorescent protein (GFP, red fluorescent protein (RFP)
  • chloramphenicol acetyltransferase chloramphenicol acetyltransferase
  • ⁇ -galactosidase ⁇ -glucuronidase
  • 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, ⁇ -galactosidase, ⁇ -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.
  • 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.
  • the rep gene expression cassette, the first complementation sequence and the cap gene expression cassette 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.
  • 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.
  • the first complementation sequence is located between the cap gene expression cassette and the rep gene expression cassette.
  • 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'.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the first homologous recombination expression cassette (SEQ ID No. 1) targeting the CNE sequence is constructed.
  • 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.
  • 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.
  • 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.
  • green fluorescent protein (GFP) or red fluorescent protein (mcherry) is used as an exogenous gene to be inserted into the AcMNPV genome.
  • 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.
  • the construct Ac96-NAE-mcherry containing the NAE sequence 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.
  • 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
  • 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.
  • 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.
  • 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.
  • Example 2 construct recombinant DNA fragments with insertion sites at orf83, orf126 and orf152 respectively, as shown in Figures 12A-F:
  • 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;
  • the packaging-deficient baculovirus vectors ⁇ CNE-Bac and ⁇ NAE-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.
  • 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 ⁇ CNE-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 ⁇ NAE-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.
  • CPE cytopathic effect
  • 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).
  • BEV-P0 the 0th generation BEV
  • 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).
  • Example 1 in the applicant's previous patent application CN112553257A, which includes the following steps:
  • ITR-GOI a recombinant transfer vector containing the ITR core element
  • the nucleotide sequence of ITR-GOI is shown in SEQ ID No. 12.
  • 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.
  • step (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
  • Example 6 The recombinant bacmid ⁇ CNE-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 ⁇ NAE-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.
  • CPE cytopathic effects
  • 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).
  • BEV-P0 0th generation BEV
  • 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
  • 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.
  • Example 1 the defective bacmid ⁇ CNE- ⁇ NAE-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 ⁇ CNE- ⁇ NAE-Bac-AAV. Please refer to Example 7 for specific steps.
  • Example 9 Purification of recombinant AAV virus particles and detection of packaging efficiency
  • Example 5 the recombinant baculovirus ⁇ CNE-Bac-AAV, ⁇ NAE-Bac-AAV and ⁇ CNE- ⁇ NAE-Bac-AAV in Example 7 and Example 8 are continued to be expanded and cultured until BEV is used.
  • 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.
  • 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.

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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

一种用于昆虫细胞中产生重组杆状病毒的组合物、方法及应用A composition, method and application for producing recombinant baculovirus in insect cells 【技术领域】【Technical field】
本发明属于基因工程技术领域，更具体地，涉及一种用于昆虫细胞中产生重组杆状病毒的组合物、方法及应用。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.
【背景技术】【Background technique】
杆状病毒是一类具有囊膜包裹的环状双链DNA病毒。目前研究较多的杆状病毒株为苜蓿银蚊夜蛾(Autographa californica)多核型多角体病毒(multiple nuclear polyhedrosis virus，MNPV)，简称AcMNPV。杆状病毒表达系统(BEVS)是一种利用昆虫杆状病毒载体感染宿主昆虫细胞生产外源蛋白的真核表达系统。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等人研究提出了BacPAK杆状病毒表达系统，该系统将lacZ基因插入到多角体病毒基因座位置，通过在orf1629和orf603基因位点各引入一个Bsu36I酶切位点构建了一种经Bsu36I酶切后线性化的复制缺陷型病毒，线性化后的病毒，由于缺失了必需基因orf1629，即使自连也无法产生有感染活性的病毒，将拯救重组DNA转移载体与线性化的病毒共转染宿主昆虫细胞，同源重组后获得重组杆状病毒。然而，由于Bsu36I酶不能100％切割病毒DNA使之线性化，重组病毒仍需要进行空斑筛选，耗时耗力。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.
随后，Lee等人研究提出了Bac-to-Bac杆状病毒表达系统，该系统在AcMNPV的多角体基因位点引入Bac人工染色体元件，该元件含有mini-F复制子和Tn7转座位点，使得杆状病毒DNA能在细菌中进行复制，同时能通过Tn7转座将外源基因插入到病毒基因组中，获得重组杆状病毒。虽然该系统不需要进行空斑筛选，但需要在细菌中进行转座重组筛选，同样耗时耗力；且通过该系统获得的重组杆状病毒在连续传代中存在不稳定的现象。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.
在2003年，zhao等人研究提出了flash-BAC系统，该系统在AcMNPV基因组中引入mini-F复制子的同时失活了必需基因orf1629，失活了必需基因orf1629的杆状病毒无法产生有感染活性的病毒，除非与拯救重组DNA转移载体发生同源重组。该系统结合了BacPAK和Bac-to-Bac的优点，只需在宿主昆虫细胞中一步重组即可获得重组杆状病毒，无需筛选。然而由于orf1629是一种与复制相关的反式元件蛋白，使得已发生同源重组的病毒DNA会提供orf1629的功能，并且由于没有发生同源重组的病毒DNA也会被包装，从而导致最终获得的重组杆状病毒不纯。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.
【发明内容】[Content of the invention]
针对现有技术的缺陷，本发明的目的在于提供一种用于昆虫细胞中产生重组杆状病毒的组合物、方法及应用，旨在解决现有利用flash-BAC系统产生重组杆状病毒过程中没有发生同源重组的病毒DNA也会被包装，且已发生同源重组的病毒DNA会提供orf1629的功能，导致重组杆状病毒不纯的问题。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.
为实现上述目的，本发明提供了一种用于昆虫细胞中产生重组杆状病毒的组合物，包含包装缺陷型杆状病毒质粒和第一拯救重组DNA，所述包装缺陷型杆状病毒质粒缺失杆状病毒基因组中的CNE序列和/或NAE序列，所述第一拯救重组DNA包含第一插入序列以及位于所述第一插入序列两侧的第一同源臂，所述第一插入序列包含第一功能片段和第 一回补序列，所述第一回补序列为所述包装缺陷型杆状病毒质粒所缺失的序列中的至少一个，所述组合物能够在昆虫细胞中发生同源重组以产生重组杆状病毒。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.
优选地，所述第一功能片段为AAV的cap基因表达盒和AAV的rep基因表达盒。Preferably, the first functional fragment is the cap gene expression cassette of AAV and the rep gene expression cassette of AAV.
优选地，所述第一插入序列按从5’至3’的顺序包含所述cap基因表达盒、所述第一回补序列和所述rep基因表达盒。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'.
优选地，所述第一插入序列按从5’至3’的顺序包含所述rep基因表达盒、所述第一回补序列和所述cap基因表达盒。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'.
优选地，所述第一回补序列位于所述cap基因表达盒与所述rep基因表达盒之间，且其两端分别靠近所述cap基因表达盒的起始端和所述rep基因表达盒的起始端。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.
优选地，所述包装缺陷型杆状病毒质粒为包含带有外源基因的AAV ITR核心表达元件的重组杆粒。Preferably, the packaging-deficient baculovirus plasmid is a recombinant bacmid containing an AAV ITR core expression element with a foreign gene.
优选地，所述重组杆粒通过杆状病毒转移载体介导的Tn7转座得到。Preferably, the recombinant bacmid is obtained by Tn7 transposition mediated by a baculovirus transfer vector.
优选地，所述第一功能片段为带有外源基因的AAV ITR核心表达元件。Preferably, the first functional fragment is an AAV ITR core expression element carrying an exogenous gene.
优选地，所述包装缺陷型杆状病毒质粒为包含AAV cap基因表达盒和AAV rep基因表达盒的重组杆粒。Preferably, the packaging-deficient baculovirus plasmid is a recombinant bacmid containing an AAV cap gene expression cassette and an AAV rep gene expression cassette.
优选地，所述重组杆粒通过杆状病毒转移载体介导的Tn7转座得到。Preferably, the recombinant bacmid is obtained by Tn7 transposition mediated by a baculovirus transfer vector.
优选地，所述第一功能片段为报告基因。Preferably, the first functional fragment is a reporter gene.
优选地，所述第一功能片段为编码治疗性基因产物的核苷酸序列。Preferably, the first functional fragment is a nucleotide sequence encoding a therapeutic gene product.
优选地，所述组合物还包含第二拯救重组DNA，所述包装缺陷型杆状病毒质粒缺失杆状病毒基因组中的CNE序列和NAE序列，所述第二拯救重组DNA包含第二插入序列以及位于所述第二插入序列两侧的第二同源臂，所述第二插入序列包含第二回补序列，所述第一回补序列为CNE序列或NAE序列，所述第二回补序列为CNE序列和NAE序列中不同于第一回补序列的序列。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.
优选地，所述第一功能片段为AAV的cap基因表达盒和AAV的rep基因表达盒，所述第二插入序列还包含第二功能片段，所述第二功能片段为带有外源基因的AAV ITR核心表达元件。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.
按照本发明的另一方面，提供了一种上述任一组合物在昆虫细胞中制备重组杆状病毒和/或重组腺相关病毒的应用。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.
按照本发明的另一方面，提供了一种昆虫细胞，包含上述任一组合物。According to another aspect of the invention, an insect cell is provided, comprising any of the above compositions.
按照本发明的另一方面，提供了一种体外生长或产生重组杆状病毒的方法，包括将上述任一组合物共转染昆虫细胞，并培养所述昆虫细胞。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.
按照本发明的另一方面，提供了一种体外生长或产生重组腺相关病毒的方法，包括将上述任一组合物共转染昆虫细胞，并培养所述昆虫细胞。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.
总体而言，通过本发明所构思的以上技术方案与现有技术相比，具有以下有益效果：Generally speaking, compared with the prior art, the above technical solution conceived by the present invention has the following beneficial effects:
本发明在flash-BAC系统的基础上进行了改进，构建包装缺陷型杆状病毒质粒，其缺失了杆状病毒基因组中的CNE序列和/或NAE序列，无法正常包装杆状病毒，除非与拯救重组DNA发生同源重组。该系统只需在宿主昆虫细胞中一步重组即可获得重组杆状病毒，无需筛选；同时插入序列可以插入到杆状病毒基因组的任意基因座，并没有插入位点的限 制，可以根据需要插入到目标位点，并且可实现从单种杆状病毒表达多种外源蛋白，灵活性更强。此外，利用该系统制备重组杆状病毒时，只有发生同源重组的病毒DNA才会被包装成为重组杆状病毒，而没有发生同源重组的病毒DNA不会被包装，因此最终获得的重组杆状病毒更纯。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.
【附图说明】[Picture description]
图1为本发明实施例1构建的打靶CNE序列的第一同源重组表达框示意图。Figure 1 is a schematic diagram of the first homologous recombination expression box targeting CNE sequence constructed in Example 1 of the present invention.
图2为本发明实施例1构建的打靶NAE序列的第二同源重组表达框示意图。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.
图3为本发明实施例1构建的打靶NAE序列的第三同源重组表达框示意图。Figure 3 is a schematic diagram of the third homologous recombination expression box targeting NAE sequence constructed in Example 1 of the present invention.
图4为本发明实施例2构建的插入位点在orf96的重组DNA片段Ac96-CNE-GFP的示意图。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.
图5为本发明实施例2构建的插入位点在orf96的重组DNA片段Ac96-NAE-mcherry的示意图。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.
图6为本发明实施例2构建的含有AAV Cap和Rep表达盒的重组DNA片段Ac96-Rep-CNE-Cap的示意图。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.
图7为本发明实施例2构建的含有AAV Cap和Rep表达盒的重组DNA片段Ac96-Rep-NAE-Cap的示意图。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.
图8为本发明实施例3利用缺陷型杆状病毒载体△CNE-Bac与重组DNA片段Ac96-CNE-GFP共转染昆虫宿主细胞后产生的绿色荧光噬斑图。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.
图9为本发明实施例3利用缺陷型杆状病毒载体△CNE-Bac与重组DNA片段Ac96-CNE-GFP共转染昆虫宿主细胞后，产生的重组杆状病毒感染细胞后表达绿色荧光的效果图。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.
图10为本发明实施例3利用缺陷型杆状病毒载体△NAE-Bac与重组DNA片段Ac96-NAE-mcherry共转染昆虫宿主细胞后产生的红色荧光噬斑图。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.
图11为本发明实施例3利用缺陷型杆状病毒载体△NAE-Bac与重组DNA片段Ac96-NAE-mcherry共转染昆虫宿主细胞后，产生的重组杆状病毒感染细胞后表达红色荧光的效果图。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.
图12A-F为本发明实施例4构建的插入位点分别在orf83、orf126和orf152的重组DNA片段的示意图。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.
图13为本发明实施例4利用缺陷型杆状病毒载体△CNE-Bac分别与重组DNA片段Ac83-CNE-GFP、Ac126-CNE-GFP和Ac152-CNE-GFP共转染昆虫宿主细胞后产生的绿色荧光噬斑图。图14为本发明实施例4利用缺陷型杆状病毒载体△CNE-Bac分别与重组DNA片段Ac83-CNE-GFP、Ac126-CNE-GFP和Ac152-CNE-GFP共转染昆虫宿主细胞后，产生的重组杆状病毒感染细胞后表达绿色荧光的效果图。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.
图15为本发明实施例4利用缺陷型杆状病毒载体△NAE-Bac分别与重组DNA片段Ac83-NAE-mcherry、Ac126-NAE-mcherry和Ac152-NAE-mcherry共转染昆虫宿主细胞后产生的红色荧光噬斑图。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.
图16为本发明实施例4利用缺陷型杆状病毒载体△NAE-Bac分别与重组DNA片段Ac83-NAE-mcherry、Ac126-NAE-mcherry和Ac152-NAE-mcherry共转染昆虫宿主细胞后， 产生的重组杆状病毒感染细胞后表达红色荧光的效果图。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.
图17为本发明实施例5利用缺陷型杆状病毒载体△CNE-Bac与重组DNA片段Ac96-Rep-CNE-Cap共转染昆虫宿主细胞后，表达VP衣壳蛋白和Rep蛋白的Western Blot检测图。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.
图18为本发明实施例5利用缺陷型杆状病毒载体△NAE-Bac与重组DNA片段Ac96-Rep-NAE-Cap共转染昆虫宿主细胞后，表达VP衣壳蛋白和Rep蛋白的Western Blot检测图。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.
图19为本发明实施例8构建的含有AAV核心表达元件ITR-GOI的重组DNA片段Ac83-ITR-NAE的示意图。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.
图20为本发明实施例9中三种重组杆状病毒转染宿主细胞后，纯化的重组AAV病毒粒子进行SDS-PAGE的银染检测图，均显示了三种衣壳蛋白VP1/VP2/VP3。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. .
【具体实施方式】【Detailed ways】
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。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.
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“及/或”包括一个或多个相关的所列项目的任意的和所有的组合。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.
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。在本发明的描述中，“若干”的含义是至少一个，例如一个，两个等，除非另有明确具体的限定。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.
正如本文所用的，表达盒是指包含引入宿主细胞时可操作连接的编码序列和调控序列的核酸构建体，分别导致RNA或多肽的转录和/或翻译。表达盒应理解为包括允许转录开始的启动子、目的基因开放阅读框和转录终止子。通常，启动子序列置于目的基因上游，与目的基因的距离与表达控制相容。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.
顺式作用元件是指与结构基因串联的特定DNA序列，是转录因子的结合位点，它们通过与转录因子结合而调控基因转录的精确起始和转录效率。顺式作用元件包括启动子、增强子、调控序列和可诱导元件等，它们的作用是参与基因表达的调控，本身不编码任何蛋白质，仅仅提供一个作用位点。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是单链DNA病毒，基因组结构简单，全长约4.7kb，其基因组中包含rep基因表达盒、cap基因表达盒和位于基因组两端的AAV反向末端重复序列(inverted terminal repeats，ITR)。这些是包装AAV病毒所必需的三个元件。Cap基因编码结构性的VP衣壳蛋白，其包含三个重叠开放阅读框，分别编码VP1、VP2、VP3三种类型的亚基。Rep基因编码Rep78、Rep68、Rep52和Rep40四个重叠的多功能蛋白，其参与AAV的复制及整合。ITR是基因组两端的125个核苷酸的回文结构，能形成一个自我互补的倒T型发卡结构，是DNA复 制起始和包装重组AAV基因组为感染性的病毒颗粒所需的顺式作用元件。AAV作为缺陷型病毒，在没有辅助病毒的存在下不能够独立复制，因此AAV只能定点整合在宿主细胞染色体中，呈潜伏状态。在辅助病毒存在的情况下，rep基因表达量增加可以将整合在宿主细胞染色体中的AAV基因组拯救出来，大量复制得到AAV DNA，单链的rAAV基因组在VP衣壳蛋白的作用下被包装成具有感染性的病毒粒子。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.
保守的非蛋白编码元件(conserved non-protein-coding element，CNE)被发现存在于所有已测序的甲型杆状病毒属(Alphabaculovirus)的基因组中，高度同源序列有154-157bp。有报道推测，位于苜蓿银纹夜蛾核型多角体病毒(Autographa californica multiple nucleopolyhedro-virus,AcMNPV)基因组ac152区域的一段富含at的CNE序列是病毒粒子生产所必需的顺式作用元件。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.
NAE序列最早被发现是在甲型杆状病毒属中存在的一种核衣壳装配必需元件，其在核衣壳装配过程中起着必不可少的作用。天然的NAE序列在甲型杆状病毒属定位于ac83基因及其同源基因中，且位于其中的近末端(CN106566829A)。ac83是与杆状病毒核衣壳装配相关的核心基因，全长2544bp，编码847个氨基酸，预测分子量为96.2kDa。敲除ac83不影响病毒基因组的复制，但会完全阻断病毒核衣壳的装配，在电子显微镜下可以观察到细胞核中出现大量中空的衣壳前体。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.
本发明提供的一种用于昆虫细胞中产生重组杆状病毒的组合物，其包含包装缺陷型杆状病毒质粒和第一拯救重组DNA，所述包装缺陷型杆状病毒质粒缺失杆状病毒基因组中的CNE序列和/或NAE序列，所述第一拯救重组DNA包含第一插入序列以及位于所述第一插入序列两侧的第一同源臂，所述第一插入序列包含第一功能片段和第一回补序列，所述第一回补序列为所述包装缺陷型杆状病毒质粒所缺失的序列中的至少一个，所述组合物能够在昆虫细胞内发生同源重组以产生重组杆状病毒。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.
需要说明的是，本发明涉及的包装缺陷型杆状病毒质粒因为缺失杆状病毒基因组中的CNE序列和/或NAE序列，导致无法正常包装出重组杆状病毒。CNE序列可以是与野生型AcMNPV CNE序列完全相同的CNE序列，也可以是来自其它杆状病毒中的CNE序列，或者是与野生型AcMNPV CNE序列享有至少50％序列同一性、至少60％序列同一性、至少70％序列同一性、至少80％序列同一性、至少85％序列同一性、至少90％序列同一性、至少95％序列同一性或更高序列同一性的人工CNE序列。同样地，NAE序列可以是与野生型AcMNPV NAE序列完全相同的NAE序列，也可以是来自其它杆状病毒中的NAE序列，或者是与野生型AcMNPV NAE序列享有至少50％序列同一性、至少60％序列同一性、至少70％序列同一性、至少80％序列同一性、至少85％序列同一性、至少90％序列同一性、至少95％序列同一性或更高序列同一性的人工NAE序列。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.
此外，当包装缺陷型杆状病毒质粒缺失杆状病毒基因组中的CNE序列时，相应的，第一回补序列为CNE序列；当包装缺陷型杆状病毒质粒缺失杆状病毒基因组中的NAE序列时，相应的，第一回补序列为NAE序列；当包装缺陷型杆状病毒质粒同时缺失杆状病毒基因组中的CNE序列和NAE序列时，相应的，第一回补序列同时包含CNE序列和NAE序列。这样使得在昆虫细胞中发生同源重组后的重组杆粒中含有正常的CNE和NAE序列，进而能够包装出重组杆状病毒；而没有发生同源重组时，杆粒上缺失CNE或NAE序列， 使得无法正常包装出重组杆状病毒。在另一个实施例中，还可以利用第二拯救重组DNA和第一拯救重组DNA共同弥补缺失序列，即当包装缺陷型杆状病毒质粒同时缺失杆状病毒基因组中的CNE序列和NAE序列时，第二拯救重组DNA包含第二插入序列以及位于第二插入序列两侧的第二同源臂，第二插入序列包含第二回补序列，第一回补序列为CNE序列或NAE序列，第二回补序列为CNE序列和NAE序列中不同于第一回补序列的序列。值得注意的是，这里的第一同源臂和第二同源臂是杆状病毒基因组上对应不同基因座的同源序列，使得第一插入序列和第二插入序列能够插入到杆状病毒基因组上的不同基因座中。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.
还需要说明的是，本发明涉及的拯救重组DNA既可以是线性DNA片段，也可以是杆状病毒转移载体，在此不做限定。另外，本发明提供的拯救重组DNA并没有插入位点的限制，当拯救重组DNA和包装缺陷型杆状病毒质粒发生同源重组时，拯救重组DNA上的插入序列能够插入到杆状病毒基因组的任意基因座，例如但不限于Ac18、Ac83、Ac96、Ac126、Ac127、Ac130、Ac152。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.
本发明中的第一功能片段可以为AAV的cap基因表达盒和AAV的rep基因表达盒，也可以为带有外源基因的AAV ITR核心表达元件(即ITR-GOI)，用于制备重组腺相关病毒。所述外源基因可以为至少一个编码感兴趣的基因(Gene of Interest，GOI)产物的核苷酸序列，所述感兴趣的基因产物可以是治疗性基因产物，具体可以是多肽、RNA分子(siRNA)或其他基因产物，例如但不限于脂蛋白酯酶、载脂蛋白、细胞因子、白细胞介素或干扰素；也可以是评估载体转化和表达的报告蛋白，例如但不限于荧光蛋白(绿色荧光蛋白GFP、红色荧光蛋白RFP)、氯霉素乙酰转移酶、β-半乳糖苷酶、β-葡萄糖醛酸酶、海肾荧光素酶、萤火虫荧光素酶或碱性磷酸酶。此外，第一功能片段还可以为报告基因，例如但不限于表达荧光蛋白(绿色荧光蛋白GFP、红色荧光蛋白RFP)、氯霉素乙酰转移酶、β-半乳糖苷酶、β-葡萄糖醛酸酶、海肾荧光素酶、萤火虫荧光素酶或碱性磷酸酶的基因序列，用于评估验证是否发生同源重组而产生重组杆状病毒。第一功能片段也可以为任何编码治疗性基因产物的核苷酸序列，例如但不限于编码药物多肽(如白细胞介素等)或病毒重组亚单位蛋白。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.
需要说明的是，在本发明实施例中，不限定cap基因表达盒与rep基因表达盒的具体位置和方向，cap基因表达盒、rep基因表达盒以及第一回补序列三部分排列方式可以有六种：1、从5’端至3’端依次排列为cap基因表达盒、rep基因表达盒和第一回补序列；2、从5’端至3’端依次排列为rep基因表达盒、cap基因表达盒和第一回补序列；3、从5’端至3’端依次排列为cap基因表达盒、第一回补序列和rep基因表达盒；4、从5’端至3’端依次排列为rep基因表达盒、第一回补序列和cap基因表达盒；5、从5’端至3’端依次排列为第一回补序列、cap基因表达盒和rep基因表达盒；6、从5’端至3’端依次排列为第一回补序列、rep基因表达盒和cap基因表达盒。cap基因表达盒与rep基因表达盒可以是同向的，也可以是反向的。cap基因表达盒在重组表达盒中的顺序既可以是从5’端至3’端的，也可以是从3’端至5’端的。同理地，rep基因表达盒在重组表达盒中的顺序既可以是从5’端至3’端的，也可以是从3’端至5’端的。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.
作为一个优选实施例，第一回补序列位于cap基因表达盒与rep基因表达盒之间，具体地，第一插入序列按从5’至3’的顺序包含cap基因表达盒、CNE序列和rep基因表达盒， 或者第一插入序列按从5’至3’的顺序包含cap基因表达盒、NAE序列和rep基因表达盒，或者第一插入序列按从5’至3’的顺序包含rep基因表达盒、CNE序列和cap基因表达盒，或者按从5’至3’的顺序包含rep基因表达盒、NAE序列和cap基因表达盒。进一步优选的，第一回补序列的两端分别靠近cap基因表达盒的起始端和rep基因表达盒的起始端，即cap基因表达盒与rep基因表达盒的方向相反，且两者的起始端相向设置并朝向第一回补序列。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.
本发明提供的一种昆虫细胞，包括上述任一种组合物。An insect cell provided by the invention includes any of the above compositions.
本发明提供的一种体外生长或产生重组杆状病毒的方法，该方法包括将上述任一种组合物共转染昆虫细胞，并培养所述昆虫细胞。然后回收可以得到重组杆状病毒。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.
本发明还提供一种体外生长或产生重组腺相关病毒的方法，该方法包括将上述组合物共转染昆虫细胞，并培养所述昆虫细胞，产生出重组腺相关病毒。需要强调的是，制备重组腺相关病毒所需的组合物中需要含有产生rAAV所必需的AAV的cap基因、rep基因和ITR核心表达元件，因此，若组合物中第一拯救重组DNA包含AAV的cap基因表达盒和AAV的rep基因表达盒，则还需要在包装缺陷型杆状病毒质粒中插入带有外源基因的AAV ITR核心表达元件，这可以通过杆状病毒转移载体介导的Tn7转座得到。若组合物中第一拯救重组DNA包含带有外源基因的AAV ITR核心表达元件，则还需要在包装缺陷型杆状病毒质粒中插入AAV的cap基因表达盒和AAV的rep基因表达盒，这可以通过杆状病毒转移载体介导的Tn7转座得到。另外，还可以通过两个拯救重组DNA来实现获得rAAV的目的，包装缺陷型杆状病毒质粒同时缺失杆状病毒基因组中的CNE序列和NAE序列，例如第一拯救重组DNA包含AAV的cap基因表达盒、AAV的rep基因表达盒和CNE序列，第二拯救重组DNA包含带有外源基因的AAV ITR核心表达元件和NAE序列。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.
以下结合具体实施例，对上述技术方案详细说明。The above technical solution will be described in detail below with reference to specific embodiments.
实施例1 构建缺失CNE序列的杆粒△CNE-Bac、缺失NAE序列的杆粒△NAE-Bac以及同时缺失CNE和NAE序列的杆粒△CNE-△NAE-BacExample 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重组是一种细菌水平的高效重组方法，可以在大肠杆菌(DH10Bac)中用来快速改造重组杆状病毒基因组。Red重组是利用λ噬菌体Red重组酶(由Exo、Beta和Gam这3种蛋白质组成)将导入细胞携带有同源臂的线性DNA片段与基因组的特定靶序列进行同源重组，从而实现目的基因的替换(Doublet等，2008，J Microbiol Methods，75(2)：359-361)。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).
首先构建打靶CNE序列的第一同源重组表达框(SEQ ID No.1)，如图1所示，该表达框从5’至3’依次包括CNE上游同源序列、氯霉素(Chol)抗性基因表达框和CNE下游同源序列；然后利用Red重组技术，将该表达框与杆粒上的CNE序列进行替换，从而获得了缺失CNE序列的杆粒△CNE-Bac。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.
同理，首先构建打靶NAE序列的第二同源重组表达框(SEQ ID No.2)，如图2所示，该表达框从5’至3’依次包括NAE上游同源序列、氯霉素(Chol)抗性基因表达框和NAE下游同源序列；然后利用Red重组技术，将该表达框与杆粒上的NAE序列进行替换，从而获得了缺失NAE序列的杆粒△NAE-Bac。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.
同样地，首先构建打靶NAE序列的第三同源重组表达框(SEQ ID No.3)，如图3所 示，该表达框从5’至3’依次包括NAE上游同源序列、庆大霉素(GM)抗性基因表达框和NAE下游同源序列；然后利用Red重组技术，将该表达框与杆粒△CNE-Bac上的NAE序列进行替换，从而获得了同时缺失CNE和NAE序列的杆粒△CNE-△NAE-Bac。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.
实施例2 构建含有CNE或NAE序列的重组DNA转移载体Example 2 Construction of recombinant DNA transfer vector containing CNE or NAE sequences
本发明各实施例采用绿色荧光蛋白(GFP)或红色荧光蛋白(mcherry)作为外源基因插入到AcMNPV基因组中。构建外源基因插入位点在AcMNPV orf96基因座的第一重组DNA片段：参见图4，该重组DNA片段从5’至3’依次包括orf96上游同源序列、CNE序列(SEQ ID No.4)、GFP表达盒和orf96下游同源序列，将上述序列通过人工直接合成或重叠延伸PCR扩增连接起来分别获得构建物Ac96-CNE-GFP。类似地，构建含有NAE序列(SEQ ID No.5)的构建物Ac96-NAE-mcherry(图5)。构建物Ac96-CNE-GFP和构建物Ac96-NAE-mcherry的核苷酸序列分别如SEQ ID No.6和SEQ ID No.7所示。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.
构建AAV rep和cap基因表达盒的插入位点在AcMNPV orf96基因座的第二重组DNA片段：参见图6，该重组DNA片段从5’至3’依次包括orf96上游同源序列、Rep基因表达盒(SEQ ID No.8)、CNE序列、Cap基因表达盒(SEQ ID No.9)和orf96下游同源序列，将上述序列通过人工直接合成或重叠延伸PCR扩增连接起来分别获得构建物Ac96-Rep-CNE-Cap。类似地，构建含有NAE序列的构建物Ac96-Rep-NAE-Cap(图7)。构建物Ac96-Rep-CNE-Cap和构建物Ac96-Rep-NAE-Cap的核苷酸序列分别如SEQ ID No.10和SEQ ID No.11所示。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.
实施例3 验证包装缺陷型杆状病毒载体与重组DNA片段共转染昆虫宿主细胞后发生同源重组产生重组杆状病毒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
本实施例将实施例1构建的包装缺陷型杆状病毒载体△CNE-Bac和△NAE-Bac与实施例2中构建的相应的重组DNA片段Ac96-CNE-GFP和Ac96-NAE-mcherry分别共转染Sf9昆虫宿主细胞，共转染96h后，利用荧光显微镜观察宿主细胞是否能够产生绿色荧光噬斑或红色荧光噬斑来判断重组杆状病毒的产生。为了进一步确认共转染确实产生了重组杆状病毒，在共转染120h后，收集Sf9细胞培养上清液，转接悬浮培养的宿主细胞，感染72h后，用荧光显微镜观察绿色荧光或红色荧光发光情况。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.
观察结果如图8至图11所示：杆状病毒载体△CNE-Bac与重组DNA片段Ac96-CNE-GFP共转染Sf9细胞96h后产生了绿色荧光噬斑(图8)，在共转染120h后，收集Sf9细胞培养上清液，转接悬浮培养的宿主细胞，感染72h后产生了大量绿色荧光(图9)；杆状病毒载体△NAE-Bac与重组DNA片段Ac96-NAE-mcherry共转染Sf9细胞96h后产生了红色荧光噬斑(图10)，在共转染120h后，收集Sf9细胞培养上清液，转接悬浮培养的宿主细胞，感染72h后产生了大量红色荧光(图11)。本实施例表明包装缺陷型杆状病毒载体△CNE-Bac或△NAE-Bac与重组DNA片段共转染昆虫宿主细胞后能发生同源重组产生重组杆状病毒，并进行外源基因的蛋白表达。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. .
实施例4 外源基因可以插入在AcMNPV基因组任意基因座，并没有插入位点的限制，可以根据需要插入到目标位点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.
首先参照实施例2构建插入位点分别在orf83、orf126和orf152的重组DNA片段，如图12A-F所示：其中构建物Ac83-CNE-GFP(图12A)、Ac126-CNE-GFP(图12B)和 Ac152-CNE-GFP((图12C)为带有CNE回补序列的插入位点分别在orf83、orf126和orf152的重组DNA片段；构建物Ac83-NAE-mcherry(图12D)、Ac126-NAE-mcherry(图12E)和Ac152-NAE-mcherry(图12F)为带有NAE回补序列的插入位点分别在orf83、orf126和orf152的重组DNA片段；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;
本实施例将实施例1中的包装缺陷型杆状病毒载体△CNE-Bac和△NAE-Bac分别与本实施例中的重组DNA片段共转染Sf9昆虫宿主细胞，共转染96h后，利用荧光显微镜观察宿主细胞是否能够产生绿色荧光噬斑或红色荧光噬斑来判断重组杆状病毒的产生。为了进一步确认共转染确实产生了重组杆状病毒，在共转染120h后，收集Sf9细胞培养上清液，转接悬浮培养的宿主细胞，感染72h后，用荧光显微镜观察绿色荧光或红色荧光发光情况。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.
观察结果如图13至图16所示：杆状病毒载体△CNE-Bac分别与重组DNA片段Ac83-CNE-GFP、Ac126-CNE-GFP和Ac152-CNE-GFP共转染Sf9细胞96h后产生了绿色荧光噬斑(图13)，在共转染120h后，收集Sf9细胞培养上清液，转接悬浮培养的宿主细胞，感染72h后产生了大量绿色荧光(图14)；杆状病毒载体△NAE-Bac分别与重组DNA片段Ac83-NAE-mcherry、Ac126-NAE-mcherry和Ac152-NAE-mcherry共转染Sf9细胞96h后产生了红色荧光噬斑(图15)，在共转染120h后，收集Sf9细胞培养上清液，转接悬浮培养的宿主细胞，感染72h后产生了大量红色荧光(图16)。本实施例显示了外源基因可以插入在AcMNPV基因组任意基因座，并没有插入位点的限制，可以根据需要插入到目标位点，并进行外源基因的表达。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.
实施例5 获得含有AAV包装必需元件Cap和Rep的重组杆状病毒及检测Cap和Rep的表达情况Example 5 Obtaining recombinant baculovirus containing Cap and Rep, essential elements for AAV packaging, and detecting the expression of Cap and Rep
将实施例1中的包装缺陷型杆状病毒载体△CNE-Bac与实施例2中的重组DNA片段Ac96-Rep-CNE-Cap共转染Sf9昆虫细胞，以及实施例1中的包装缺陷型杆状病毒载体△NAE-Bac与实施例2中的重组DNA片段Ac96-Rep-NAE-Cap分别共转染Sf9昆虫细胞，制备重组杆状病毒BEV。转染后的Sf9昆虫细胞成功产生BEV，大量复制增殖的BEV进一步感染导致Sf9细胞发生明显的细胞病变效应(cytopathic effect，CPE)。收集发生CPE的Sf9细胞培养上清液，其中含有大量BEV，即为第0代BEV(BEV-P0)，同时收集含有大量rAAV的Sf9细胞。将制备得到的BEV-P0以感染复数(MOI＝1)感染悬浮培养的Sf9细胞，感染72h后，细胞活性下降至50％以下，将细胞培养液1000g离心5min，分别收集培养上清和细胞沉淀，上清液标记为第1代BEV(BEV-P1)。继续扩大培养，将制备得到的BEV-P1以感染复数(MOI＝1)感染悬浮培养的Sf9细胞，感染72h后，细胞活性下降至50％以下，将细胞培养液1000g离心5min，收集细胞沉淀进行Western Blot检测VP蛋白(VP1、VP2、VP3)和Rep蛋白(Rep78、Rep52)的表达情况。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).
结果如图17和图18所示：杆状病毒载体△CNE-Bac与重组DNA片段Ac96-Rep-CNE-Cap共转染Sf9昆虫细胞后产生了重组杆状病毒，并成功表达出VP蛋白和Rep蛋白(图17)；杆状病毒载体△NAE-Bac与重组DNA片段Ac96-Rep-NAE-Cap共转染Sf9昆虫细胞后产生了重组杆状病毒，并成功表达出VP蛋白和Rep蛋白(图18)。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).
实施例6 构建含有AAV核心表达元件(ITR-GOI)的重组杆状病毒载体Example 6 Construction of recombinant baculovirus vector containing AAV core expression element (ITR-GOI)
本实施例构建用于在昆虫细胞中生产AAV病毒的重组AAV杆粒的方法参照本发明申请人此前申请专利CN112553257A中的实施例1，包括如下步骤：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)构建包含ITR核心元件(ITR-GOI)的重组转移载体。ITR-GOI的核苷酸序列如SEQ ID No.12所示。本实施例中ITR核心元件中的GOI采用了红色荧光蛋白mcherry基因表达盒，即由miniEf1a启动子控制mcherry表达，便于检测rAAV的活性，将ITR和红色荧光蛋白表达盒构建到转移载体pFastDual上。(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)用上述步骤(1)中构建的重组转移载体转化含有△CNE-Bac或△NAE-Bac杆粒的感受态细胞，利用Tn7重组将ITR-GOI插入到△CNE-Bac或△NAE-Bac杆粒的Tn7位点上，最终得到包含生产rAAV所必需的ITR核心元件的重组杆状病毒质粒，编号分别为△CNE-Bac-Tn7-ITR和△NAE-Bac-Tn7-ITR。(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.
实施例7 利用△CNE-Bac-Tn7-ITR或△NAE-Bac-Tn7-ITR缺陷型杆粒制备含有AAV包装必需元件Cap、Rep和ITR-GOI的AAV重组杆状病毒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
将实施例6中制备的重组杆粒△CNE-Bac-Tn7-ITR与实施例2中制备的重组DNA片段Ac97-Rep-CNE-Cap，以及施例6中制备的重组杆粒△NAE-Bac-Tn7-ITR与实施例2中制备的重组DNA片段Ac97-Rep-NAE-Cap分别共转染宿主昆虫细胞系培养获得AAV重组杆状病毒，编号分别为△CNE-Bac-AAV和△NAE-Bac-AAV，具体步骤如下：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:
抽提上述重组杆粒和转移载体DNA共转染Sf9昆虫细胞，制备重组杆状病毒BEV和rAAV。共转染后的Sf9昆虫细胞成功产生BEV，大量复制增殖的BEV进一步感染导致Sf9细胞发生明显的细胞病变效应(CPE)。收集发生CPE的Sf9细胞培养上清液，其中含有大量BEV，即为第0代BEV(BEV-P0)，同时收集含有大量rAAV的Sf9细胞。将制备得到的BEV-P0以感染复数(MOI＝1)感染悬浮培养的Sf9细胞，感染72h后，细胞活性下降至50％以下，将细胞培养液1000g离心5min，分别收集培养上清和细胞沉淀，上清液标记为第1代BEV(BEV-P1)，细胞则标记为用BEV-P0包装的rAAV。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.
实施例8 利用△CNE-△NAE-Bac缺陷型杆粒制备含有AAV包装必需元件Cap、Rep和ITR-GOI的AAV重组杆状病毒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
首先构建AAV ITR核心表达元件(ITR-GOI)的插入位点在AcMNPV orf83基因座的重组DNA片段：参见图19，该重组DNA片段从5’至3’依次包括orf83上游同源序列、ITR-GOI序列、NAE序列和orf83下游同源序列，将上述序列通过人工直接合成或重叠延伸PCR扩增连接起来获得构建物Ac83-ITR-NAE。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.
然后将实施例1中制备的缺陷型杆粒△CNE-△NAE-Bac与实施例2中制备的重组DNA片段Ac97-Rep-CNE-Cap，以及本实施例中制备的重组DNA片段Ac83-ITR-NAE共转染宿主昆虫细胞系培养获得AAV重组杆状病毒，编号为△CNE-△NAE-Bac-AAV，具体步骤参照实施例7。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.
实施例9 重组AAV病毒粒子的纯化及其包装率的检测Example 9 Purification of recombinant AAV virus particles and detection of packaging efficiency
按照实施例5的操作将实施例7和实施例8中的重组杆状病毒△CNE-Bac-AAV、△NAE-Bac-AAV和△CNE-△NAE-Bac-AAV继续扩大培养，直至用BEV-P2的种毒按照感染复数(MOI＝1)感染悬浮培养的Sf9细胞进行rAAV的包装，包装体积为300mL～400mL。感染3天后监测细胞活性，活性低于50％，分别离心收获细胞沉淀和上清，将收获的细胞 沉淀和上清分别纯化，细胞反复冻融3次裂解，5000rpm离心10min收集上清，在上清中加入核酸酶(Benzonase)37℃水浴处理60min，处理后5000rpm离心10min。收集的细胞裂解液和收集的上清液PEG沉淀，重悬后用碘克沙醇密度梯度离心分离纯化(方法参见Aslanidi等，2009，Proc.Natl.Acad.Sci.USA,206:5059-5064)。最终纯化的成品病毒用80μL～190μL PBS重悬，取10μL纯化出的成品病毒跑SDS-PAGE胶，银染。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.
SDS-PAGE胶图如图20所示：其中，泳道1为重组杆状病毒△CNE-Bac-AAV感染宿主细胞后，纯化的重组AAV病毒粒子进行SDS-PAGE的银染检测图，显示了三种衣壳蛋白VP1/VP2/VP3；泳道2为重组杆状病毒△NAE-Bac-AAV感染宿主细胞后，纯化的重组AAV病毒粒子进行SDS-PAGE的银染检测图，显示了三种衣壳蛋白VP1/VP2/VP3；泳道3为重组杆状病毒△CNE-△NAE-Bac-AAV感染宿主细胞后，纯化的重组AAV病毒粒子进行SDS-PAGE的银染检测图，显示了三种衣壳蛋白VP1/VP2/VP3。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.
本实施例还采用Q-PCR检测收获的rAAV病毒的包装率，rAAV包装率的检测使用靶向ITR序列的一对引物(Q-ITR-F:GGAACCCCTAGTGATGGAGTT和Q-ITR-R:CGGCCTCAGTGAGCGA)。检测结果如表1。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.
表1 重组杆状病毒生产的rAAV病毒粒子的包装率检测结果表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
本实施例显示了利用本发明提供的缺陷型杆状病毒载体及重组DNA片段，可直接在昆虫宿主细胞中重组，制备含有AAV包装必须元件Cap、Rep和ITR-GOI的AAV重组杆状病毒，并成功产生了AAV病毒粒子。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.
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。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 (18)

  1. 一种用于昆虫细胞中产生重组杆状病毒的组合物，其特征在于：包含包装缺陷型杆状病毒质粒和第一拯救重组DNA，所述包装缺陷型杆状病毒质粒缺失杆状病毒基因组中的CNE序列和/或NAE序列，所述第一拯救重组DNA包含第一插入序列以及位于所述第一插入序列两侧的第一同源臂，所述第一插入序列包含第一功能片段和第一回补序列，所述第一回补序列为所述包装缺陷型杆状病毒质粒所缺失的序列中的至少一个，所述组合物能够在昆虫细胞中发生同源重组以产生重组杆状病毒。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.
  2. 根据权利要求1所述的组合物，其特征在于：所述第一功能片段为AAV的cap基因表达盒和AAV的rep基因表达盒。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.
  3. 根据权利要求2所述的组合物，其特征在于：所述第一插入序列按从5’至3’的顺序包含所述cap基因表达盒、所述第一回补序列和所述rep基因表达盒。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.
  4. 根据权利要求2所述的组合物，其特征在于：所述第一插入序列按从5’至3’的顺序包含所述rep基因表达盒、所述第一回补序列和所述cap基因表达盒。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.
  5. 根据权利要求2所述的组合物，其特征在于：所述第一回补序列位于所述cap基因表达盒与所述rep基因表达盒之间，且其两端分别靠近所述cap基因表达盒的起始端和所述rep基因表达盒的起始端。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.
  6. 根据权利要求2所述的组合物，其特征在于：所述包装缺陷型杆状病毒质粒为包含带有外源基因的AAV ITR核心表达元件的重组杆粒。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.
  7. 根据权利要求6所述的组合物，其特征在于：所述重组杆粒通过杆状病毒转移载体介导的Tn7转座得到。The composition according to claim 6, wherein the recombinant bacmid is obtained by Tn7 transposition mediated by a baculovirus transfer vector.
  8. 根据权利要求1所述的组合物，其特征在于：所述第一功能片段为带有外源基因的AAV ITR核心表达元件。The composition according to claim 1, characterized in that: the first functional fragment is an AAV ITR core expression element with an exogenous gene.
  9. 根据权利要求8所述的组合物，其特征在于：所述包装缺陷型杆状病毒质粒为包含AAV cap基因表达盒和AAV rep基因表达盒的重组杆粒。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.
  10. 根据权利要求9所述的组合物，其特征在于：所述重组杆粒通过杆状病毒转移载体介导的Tn7转座得到。The composition according to claim 9, wherein the recombinant bacmid is obtained by Tn7 transposition mediated by a baculovirus transfer vector.
  11. 根据权利要求1所述的组合物，其特征在于：所述第一功能片段为报告基因。The composition according to claim 1, wherein the first functional fragment is a reporter gene.
  12. 根据权利要求1所述的组合物，其特征在于：所述第一功能片段为编码治疗性基因产物的核苷酸序列。The composition according to claim 1, wherein the first functional fragment is a nucleotide sequence encoding a therapeutic gene product.
  13. 根据权利要求1所述的组合物，其特征在于：还包含第二拯救重组DNA，所述包装缺陷型杆状病毒质粒缺失杆状病毒基因组中的CNE序列和NAE序列，所述第二拯救重组DNA包含第二插入序列以及位于所述第二插入序列两侧的第二同源臂，所述第二插入序列包含第二回补序列，所述第一回补序列为CNE序列或NAE序列，所述第二回补序列为CNE序列和NAE序列中不同于第一回补序列的序列。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.
  14. 根据权利要求13所述的组合物，其特征在于：所述第一功能片段为AAV的cap基因表达盒和AAV的rep基因表达盒，所述第二插入序列还包含第二功能片段，所述第二功能片段为带有外源基因的AAV ITR核心表达元件。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.
  15. 一种权利要求1-14任一所述的组合物在昆虫细胞中制备重组杆状病毒和/或重组腺相关病毒的应用。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.
  16. 一种昆虫细胞，其特征在于：包含权利要求1-14任一所述的组合物。An insect cell, characterized in that it contains the composition according to any one of claims 1-14.
  17. 一种体外生长或产生重组杆状病毒的方法，其特征在于：包括将权利要求1-14任一所述的组合物共转染昆虫细胞，并培养所述昆虫细胞。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.
  18. 一种体外生长或产生重组腺相关病毒的方法，其特征在于：包括将权利要求6、7、9、10和14中任一所述的组合物共转染昆虫细胞，并培养所述昆虫细胞。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 .
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