WO2023092643A1 - 一种包含重叠开放阅读框的基因的表达盒及其在昆虫细胞中的应用 - Google Patents

一种包含重叠开放阅读框的基因的表达盒及其在昆虫细胞中的应用 Download PDF

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WO2023092643A1
WO2023092643A1 PCT/CN2021/135642 CN2021135642W WO2023092643A1 WO 2023092643 A1 WO2023092643 A1 WO 2023092643A1 CN 2021135642 W CN2021135642 W CN 2021135642W WO 2023092643 A1 WO2023092643 A1 WO 2023092643A1
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expression cassette
intron
gene
aav
open reading
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PCT/CN2021/135642
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French (fr)
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肖何
何晓斌
黄刚
胡颖
杜亮
王梦蝶
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劲帆生物医药科技（武汉）有限公司
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Priority to JP2022576350A priority Critical patent/JP2024500194A/ja
Priority to US18/016,684 priority patent/US20230383310A1/en
Priority to EP21952112.7A priority patent/EP4209594A1/en
Priority to AU2021456513A priority patent/AU2021456513A1/en
Publication of WO2023092643A1 publication Critical patent/WO2023092643A1/zh

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Definitions

  • the invention belongs to the technical field of genetic engineering, and more specifically relates to a gene expression cassette containing overlapping open reading frames and its application in insect cells.
  • Adeno-associated virus also known as adeno-associated virus, belongs to the family Parvoviridae and is the most simple single-stranded DNA defective virus found so far. virus) involved in replication.
  • Recombinant adeno-associated virus rAAV is one of the most promising vectors in the field of gene therapy due to its wide host range, low immunogenicity, high safety, and ability to mediate long-term stable expression of foreign genes in animals. .
  • rAAV adeno-associated virus
  • the cap gene in the adeno-associated virus genome encodes the viral VP capsid protein, which contains three overlapping open reading frames, which encode three types of subunits, VP1, VP2, and VP3.
  • the stoichiometric ratio is about 1:1:10;
  • the rep gene encodes four overlapping multifunctional proteins, Rep78, Rep68, Rep52 and Rep40, which are mainly involved in the replication of AAV.
  • the normal stoichiometric expression of the three AAV capsid proteins can be regulated by relying on the alternate use of the two splice acceptor sequences of the intron and the use of the suboptimal start codon ACG of VP2, thereby packaging the Normal recombinant adeno-associated virus.
  • Urabe et al. developed an insect cell production system, replacing the start codon AUG of VP1 with a suboptimal start codon ACG to construct a single polycistronic mRNA, the polycistronic mRNA All three VP proteins of AAV2 are expressed without splicing (Urabe et al., 2002, Hum Gene Ther, 13:1935-1943).
  • the transformation method of Urabe et al. is not suitable for all serotypes. Good infectivity.
  • a different suboptimal start codon CTG is used to enhance the expression of VP1.
  • Urabe et al. constructed two independent expression cassettes of Rep78 and Rep52, and inserted them into the same baculovirus vector. Since the low expression of Rep78 is conducive to the packaging of AAV, Rep52 uses the Polh promoter, and Rep78 uses the ⁇ IE1 promoter with relatively weak activity relative to the Polh promoter (Urabe et al., 2002, Hum Gene Ther, 13:1935-1943 ).
  • the AAV Rep protein expression method developed by Urabe et al. has the problem of unstable passage of baculovirus vectors.
  • Chinese patent CN103849629A discloses a method for producing AAV in insect cells. AAVRep78 The translation start codon of the protein was replaced by ACG. Although this method can realize the expression of Rep78 and Rep52 proteins in the same reading frame, it lacks the regulation effect on the relative expression of Rep78 and Rep52 proteins.
  • the object of the present invention is to provide a gene expression cassette comprising overlapping open reading frames and its application in insect cells, which can achieve stable and large-scale production in insect cells with a high packaging rate Recombinant adeno-associated virus with infectious activity, and suitable for various serotypes.
  • the present invention provides an expression cassette for expressing genes comprising overlapping open reading frames in insect cells, which comprises from 5' to 3', operably linked:
  • a promoter capable of driving transcription in insect cells A promoter capable of driving transcription in insect cells
  • the artificially constructed sequence comprises a natural or artificially modified first intron and a second intron that have splicing activity in insect cells, and the first intron and the second intron Sharing the 5' part or the 3' part, the artificially constructed sequence does not contain a translation initiation codon;
  • a translation initiation codon AUG can be formed in the artificially constructed sequence, thereby realizing the regulation of the Translational expression of distinct protein-coding genes in overlapping open reading frames.
  • the first intron or the second intron is located between any adjacent two nucleotides in the ATG.
  • the artificially constructed sequence comprises from 5' to 3', operably linked: the 5' part of the first intron, adenine nucleotide (A), the second intron The 5' part, common 3' part, thymine nucleotide (T) and guanine nucleotide (G);
  • the artificially constructed sequence comprises from 5' to 3', operably linked: the 5' part of the first intron, adenine nucleotide (A), thymine nucleotide (T), The 5' part of the second intron, the common 3' part and the guanine nucleotide (G).
  • the 5'-terminal nucleotides of the first intron and the second intron are both GTNN, and the 3'-terminal nucleosides of the first intron and the second intron are
  • the acids are all NNAG, where N is any one of the four nucleotides A, T, C, and G.
  • the gene comprising overlapping open reading frames is the cap gene of AAV or the rep gene of AAV.
  • the promoter is a polh promoter or a p10 promoter.
  • the gene comprising overlapping open reading frames is the cap gene of AAV, and the promoter is the p10 promoter.
  • the gene comprising overlapping open reading frames is the rep gene of AAV, and the promoter is the polh promoter.
  • nucleic acid molecule comprising a first expression cassette, which is the above-mentioned expression cassette.
  • the nucleic acid molecule further comprises a second expression cassette, the second expression cassette is the above expression cassette, and the gene expressed by the second expression cassette is different from the gene expressed by the first expression cassette.
  • said second expression cassette is in an antisense orientation relative to said first expression cassette.
  • said second expression cassette is in a sense orientation relative to said first expression cassette.
  • the nucleic acid molecule further comprises an exogenous gene and AAV inverted terminal repeats located at both ends of the exogenous gene.
  • the exogenous gene is a reporter gene
  • the reporter gene is a gene encoding chloramphenicol acetyltransferase, a gene encoding ⁇ -galactosidase, a gene encoding ⁇ -glucuronidase, a gene encoding Renilla luciferase At least one of gene, alkaline phosphatase encoding gene, firefly luciferase encoding gene, green fluorescent protein encoding gene and red fluorescent protein encoding gene.
  • the exogenous gene is a gene encoding a pharmaceutical polypeptide or a gene encoding an antigenic protein used to prepare a vaccine for animals or humans.
  • a vector comprising the above expression cassette is provided.
  • the carrier is an insect cytocompatible carrier.
  • the vector is at least one of a plasmid and a virus.
  • it provides the use of the above-mentioned vector to prepare recombinant adeno-associated virus in insect cells.
  • the application of the above-mentioned vector in the preparation of AAV capsid in vitro and the gene comprising the overlapping open reading frame is the cap gene of AAV.
  • an insect cell comprising the above expression cassette is provided.
  • said expression cassette is integrated into the genome of said insect cell.
  • the insect cells are Spodoptera frugiperda cells, Trichoplusia nigra cells, Drosophila cells or mosquito cells.
  • a cell culture comprising the above-mentioned insect cells and a culture medium.
  • the culture medium contains AAV genome.
  • a recombinant adeno-associated virus particle which is obtained by culturing the above-mentioned insect cells under conditions capable of producing recombinant adeno-associated virus particles, and then recovering them.
  • the expression cassette of the present invention can be used to express in insect cells a variety of polypeptides encoded by genes comprising overlapping open reading frames, such as VP proteins (VP1, VP2, VP3) or Rep proteins (Rep78, Rep52) of recombinant adeno-associated viruses ), the present invention does not need to change the original translation start codon ATG of the VP1 protein coding sequence or the Rep78 protein coding sequence in the wild-type AAV, by inserting two introns downstream of a promoter and near the start codon ATG, utilizing The splicing of these two introns can perform alternative splicing on the post-transcriptional mRNA to obtain two or more mRNAs in appropriate proportions, which can be translated into VP1, VP2/VP3 proteins or Rep78, Rep52 proteins, respectively.
  • VP proteins VP1, VP2, VP3
  • Rep proteins Rep78, Rep52
  • the present invention uses the alternative splicing of two introns to realize the regulatory strategy for overlapping gene expression, which can more effectively control the stoichiometric ratio of VP1, VP2 and VP3 proteins and Relative expression of Rep78/Rep52 for stable and large-scale production of recombinant adeno-associated virus in insect cells.
  • the present invention uses an intron alternative splicing strategy to realize the production of recombinant adeno-associated virus in insect cells, and is applicable to various AAV serotypes, making the preparation of rAAV more flexible and wider in application.
  • the cap gene, rep gene and ITR core expression element with foreign gene of AAV are constructed in the same recombinant baculovirus vector, and the vector is used to transfect insect host cells, which can stably and high-yield produce recombinant AAV virions.
  • Fig. 1 is a schematic diagram of cap gene and rep gene expression regulation in wild-type AAV of the present invention.
  • Figure 2 is a schematic diagram of cap gene expression cassette expression regulation in Example 1 of the present invention, wherein, content A is a schematic diagram of cap gene expression cassette DNA chain; content B is the first intron and the second intron in the process of post-transcriptional processing Schematic diagram of translation and expression of VP2 and VP3 proteins when no splicing occurs; content C is a schematic diagram of translation and expression of VP1 protein after the second intron is spliced during post-transcriptional processing; content D is the schematic diagram of the first intron during post-transcriptional processing Schematic diagram of the translation and expression of VP2 and VP3 proteins after splicing.
  • Figure 3 is a schematic diagram of the expression regulation of the rep gene expression cassette in Example 2 of the present invention, wherein, content A is a schematic diagram of the DNA chain of the rep gene expression cassette; content B is the first intron and the second intron in the post-transcriptional processing process Schematic diagram of Rep52 protein translation and expression when no splicing occurs; content C is a schematic diagram of Rep78 protein translation and expression after the second intron is spliced during post-transcriptional processing; content D is the first intron during post-transcriptional processing Schematic representation of the translational expression of Rep52 protein after splicing.
  • content A is a schematic diagram of the DNA chain of the rep gene expression cassette
  • content B is the first intron and the second intron in the post-transcriptional processing process Schematic diagram of Rep52 protein translation and expression when no splicing occurs
  • content C is a schematic diagram of Rep78 protein translation and expression after the second intron is spliced during post-transcriptional processing
  • content D is the first intron during post
  • Fig. 4 is a Western Blot detection diagram of VP protein expressed by the recombinant baculovirus vector Ac1 containing the cap gene expression cassette in Example 3 of the present invention and the recombinant baculovirus vector Ac0 prepared in the comparative example.
  • Fig. 5 is a Western Blot detection diagram of the expression of Rep protein by the recombinant baculovirus vector Ac2 containing the rep gene expression cassette in Example 3 of the present invention.
  • Figure 6 is a silver staining diagram of SDS-PAGE of the purified recombinant AAV virions after the recombinant AAV bacmids CRI-0 and CRI-1 are transfected into host cells in Example 6 of the present invention, showing three capsid proteins VP1 /VP2/VP3.
  • Fig. 7 is a diagram showing the effect of purified recombinant AAV virus particles infecting 293T cells after the recombinant AAV bacmids CRI-0 and CRI-1 are transfected into host cells in Example 6 of the present invention.
  • Fig. 8 is the result of SDS-PAGE of purified recombinant AAV virions after recombinant AAV bacmids CRI-2 (A), CRI-3 (B) and CRI-4 (C) transfect host cells in Example 7 of the present invention
  • Fig. 9 is a diagram showing the effect of purified recombinant AAV virus particles infecting 293T cells after recombinant AAV bacmids CRI-2, CRI-3 and CRI-4 are transfected into host cells in Example 7 of the present invention.
  • operably linked refers to the linkage of polynucleotide (or polypeptide) sequences in a functional relationship. Two nucleotide sequences are “operably linked” when they are placed into a functional relationship.
  • a transcriptional regulatory sequence eg, a promoter
  • a gene coding sequence if it affects the transcription of the gene coding sequence.
  • expression cassette refers to a nucleic acid construct comprising operably linked coding and regulatory sequences that when introduced into a host cell result 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.
  • Open Reading Frame (Open Reading Frame, ORF) is the normal nucleotide sequence of a structural gene, with the potential to encode a protein or polypeptide, starting from a start codon and ending with a stop codon, without interrupting translation stop codon.
  • ORF Open Reading Frame
  • the ribosome starts to translate from the start codon, synthesizes a polypeptide chain along the mRNA sequence and continues to extend it, and when it encounters a stop codon, the extension reaction of the polypeptide chain is terminated.
  • vector refers to a nucleic acid molecule designed to transport, transfer and/or store genetic material, as well as express genetic material and/or integrate genetic material into the chromosomal DNA of a host cell, such as plasmid vectors, cosmid vectors, artificial chromosomes , phage vectors and other viral vectors.
  • a vector usually consists of at least three basic units, namely a replication source, a selectable marker and a multiple cloning site.
  • intron also known as an intervening sequence, refers to a non-coding segment in a gene or mRNA molecule, and is an intervening sequence in eukaryotic cell DNA. Intron sequences are transcribed in the pre-mRNA, removed by splicing, and finally absent from the mature mRNA molecule. According to whether the splicing process is spontaneous or processed by the spliceosome, introns are divided into self-splicing introns and splice introns. Self-splicing introns are a special kind of introns, which are ribozymes that can be excised by themselves to leave mRNA.
  • the introns involved in the present invention are splice introns, and the splicing of such introns requires the help of splice bodies.
  • the spliceosome is a ribonucleoprotein complex dynamically composed of small nuclear RNA (snRNA) and protein factors. The spliceosome recognizes the splicing site of the pre-mRNA and catalyzes the splicing reaction, completely cuts out the intron, and the upstream and downstream RNA The sequence is then reconnected.
  • AAV serotype Since the discovery of AAV, more than 100 AAV serotypes or mutants have been isolated from adenoviruses, humans or primates, and some mammals, mainly AAV1-9 . The main difference between rAAV vectors of different serotypes is the capsid protein. Different AAV serotypes have certain differences in infection efficiency and tissue specificity.
  • AAV is a single-stranded DNA virus with a simple genome structure and a total length of about 4.7kb.
  • its genome contains rep gene expression cassettes and cap gene expression cassettes and AAV inverted terminal repeats (inverted terminal repeats, ITR) located at both ends of the genome.
  • ITR inverted terminal repeats
  • ITR is a palindromic structure of 125 nucleotides at both ends of the genome, which can form a self-complementary inverted T-shaped hairpin structure, and is a cis-acting element required for DNA replication initiation and packaging of recombinant AAV genomes into infectious virus particles .
  • the Cap gene encodes a structural VP capsid protein, which contains three overlapping open reading frames, which encode three types of subunits, VP1, VP2, and VP3, respectively. VP1, VP2, and VP3 contain different start codons and share one Stop codon, VP1 and VP2 share the VP3 sequence.
  • the N-terminus of VP1 has a conserved phospholipase A2 sequence, which is related to the escape of the virus from the body and is critical for its infectivity; the VP2 protein is not indispensable for assembly or infection; the core of the VP3 protein consists of a conserved ⁇ -Barrel motif composition determines differences in receptors for different serotypes of AAV to interact with host cells.
  • the correct ratio of the three proteins in wild-type AAV is 3:3:54, which is approximately 1:1:10.
  • the Rep gene encodes four overlapping multifunctional proteins, Rep78, Rep68, Rep52, and Rep40.
  • the Rep78 and Rep68 proteins are involved in the replication and integration of AAV, and can bind to specific sequences in the ITR; the Rep52 and Rep40 proteins have the functions of helicase and ATPase Activity, while participating in the replication of the single-stranded genome, it is also involved in the assembly of the virus. Whether in mammalian cells or in insect cells, unspliced mRNAs encoding Rep78 and Rep52 proteins are sufficient to meet the requirements for the production of rAAV.
  • the nucleic acid molecules mentioned in the present invention may be DNA molecules or RNA molecules.
  • RNA sequences are basically similar to DNA sequences or have a certain degree of sequence identity, and thymine (T) in DNA sequences can be considered equivalent to uracil (U) in RNA sequences.
  • the intron mentioned in the present invention can be a natural intron or an artificially modified intron, and it has splicing activity in insect cells, but its source is not limited to insect cells.
  • the present invention provides an expression cassette for expressing genes comprising overlapping open reading frames in insect cells comprising operably linked from 5' to 3':
  • a promoter capable of driving transcription in insect cells A promoter capable of driving transcription in insect cells
  • the artificially constructed sequence comprises a natural or artificially modified first intron and a second intron that have splicing activity in insect cells, and the first intron and the second intron Sharing the 5' part or the 3' part, the artificially constructed sequence does not contain a translation initiation codon;
  • a translation initiation codon AUG can be formed in the artificially constructed sequence, thereby realizing the regulation of the Translational expression of distinct protein-coding genes in overlapping open reading frames.
  • the first intron or the second intron is located between any two adjacent nucleotides in the ATG.
  • the arrangement order of the first intron and the second intron is not particularly limited. Take the first intron located upstream of the second intron as an example: when the first intron is located at any two adjacent When the part between nucleotides and the second intron that is not shared with the first intron (3' part) is located downstream of G in the ATG, during post-transcriptional processing, the first intron Splicing, which can form the translation initiation codon AUG upstream of the overlapping open reading frame; when the second intron is located between any adjacent two nucleotides in ATG, the first intron does not and the second When the part shared by introns (5' part) is located upstream of A in this ATG, the translation initiation codon can be formed upstream of the overlapping open reading frame by the splicing of the second intron during post-transcriptional processing Sub-AUG.
  • the expression cassette containing two introns can be used for correct expression of AAVVP capsid protein or dominant expression of Rep protein in insect cells by utilizing intron alternative splicing, wherein, the overlap in the cap gene
  • the open reading frames encode the AAV capsid proteins VP1, VP2 and VP3; the overlapping open reading frames in the rep gene encode the Rep78 and Rep52 proteins.
  • an expression cassette comprising two introns can be used to express various simian virus 40 (SV40) VP capsid proteins in insect cells using alternative intron splicing.
  • SV40 is a double-stranded DNA virus with a circular genome with a covalently closed loop of 5.2 kb. It also contains three capsid proteins VP1, VP2 and VP3.
  • the VP3 protein is a truncated form of the VP2 protein.
  • VP3 and VP2 share a stop codon.
  • the 5' part of the VP1 coding sequence overlaps with the 3' part of the VP2 and VP3 coding sequences, but they do not have the same open reading frame.
  • the expression of these VP proteins is regulated by the intron splicing machinery.
  • the expression cassette of the present invention is used to express the SV40 capsid protein in insect cells, the ORF that only lacks the first translation initiation codon in the expression cassette is the VP protein coding sequence that only lacks the translation initiation codon ATG of the VP2 protein, The relative expression control of VP2 and VP3 proteins can be realized by using the intron splicing strategy of the present invention.
  • the gene comprising the overlapping open reading frame is the cap gene of AAV, and the overlapping open reading frame that only lacks the first translation initiation codon is the VP that only lacks the translation initiation codon ATG of the VP1 protein
  • the artificially constructed sequence comprises from 5' to 3', operably linked: the 5' part of the first intron, adenine nucleotide (A), the second intron the 5' portion of the first intron, the common 3' portion, thymine nucleotide (T) and guanine nucleotide (G); or the 5' portion of the first intron, adenine nucleotide, thymine nucleotides, the 5' portion of the second intron, the common 3' portion and guanine nucleotides.
  • the present invention mutates the intron splice site at the N-terminal of the wild-type AAVVP1 protein coding sequence, so that the splice body in insect cells cannot recognize the splice site, and the 5' part of the first intron and the common 3' part are formed
  • the complete first intron splice site, the 5' part of the second intron and the common 3' part form the complete second intron splice site, as shown in Figure 2, during post-transcriptional processing, If the spliceosome in insect cells recognizes the first intron splice site and catalyzes the splicing reaction, a complete AUG-start site cannot be formed at the front of the mRNA, and the ribosome recognizes the start of the VP2 protein from 5' to 3' When the start codon is used, the VP2 protein can be translated and expressed.
  • the VP2 start codon is the suboptimal codon ACG, it will cause ribosome scanning leaks, so the VP3 protein can be translated and expressed; if the spliceosome in insect cells recognizes the second intron The sub-splicing site and catalyze the splicing reaction, after splicing to form a complete AUG-initiation site, the mRNA is translated from the first AUG, and the VP1 protein is expressed; if the splicing body in insect cells neither splices the first intron The splicing site and the second intron splicing site are not spliced, the 5' end of the VP1 protein coding sequence has no start codon, and the VP2 and VP3 proteins are translated and expressed.
  • the relative expression of capsid proteins VP1, VP2 and VP3 is controlled by splicing with different probabilities of different introns.
  • the gene comprising the overlapping open reading frame is the rep gene of AAV, and the overlapping open reading frame that only lacks the first translation initiation codon is the Rep that only lacks the translation initiation codon ATG of the Rep78 protein
  • a protein coding sequence the artificially constructed sequence comprising from 5' to 3', operably linked: the 5' part of the first intron, adenine nucleotides, 5' of the second intron ' part, common 3' part, thymine nucleotide and guanine nucleotide; or the 5' part of said first intron, adenine nucleotide, thymine nucleotide, said second intron
  • the present invention mutates one or more possible translation initiation sites upstream of the translation initiation codon of the Rep52 protein in the coding sequence of the wild-type AAVRep78 protein, so that ribosomes in insect cells cannot recognize these sites, and the first containing The 5' part of the intron and the common 3' part form the complete first intron splice site, and the 5' part of the second intron and the common 3' part form the complete second intron splice site, as shown in the figure 3, during post-transcriptional processing, if the spliceosome in insect cells recognizes the first intronic splice site and catalyzes the splicing reaction, a complete AUG-initiation site cannot be formed at the front end of the mRNA, and the ribosome When the start codon of the Rep52 protein is recognized from 5' to 3', the Rep52 protein can be translated and expressed; if the splicing body in insect cells recognizes the second intron splicing site and catalyzes the
  • both the 5' terminal nucleotides of the first intron and the second intron are GTNN, and the nucleotides of the first intron and the second intron are The 3' terminal nucleotides are all NNAG, where N represents any one of the four nucleotides A, T, C, and G.
  • This can regulate the translation and expression of VP1, VP2 and VP3 proteins to be closer to the stoichiometric ratio, which is conducive to packaging into a suitable capsid; similarly, the translation and expression of Rep78 protein is lower than that of Rep52 protein, which is conducive to higher vector production. .
  • the nucleotide sequence of the artificially constructed sequence is the sequence shown in SEQ ID No.2.
  • SEQ ID No.2 Those skilled in the art know that variations of the intron sequence can be used in the technical solution of the present invention.
  • a sequence having similarity to the nucleotide sequence of the intron of the invention may be used, for example, the 5' end of the 5' portion of the first intron has at least 40% or 50% or 60% similarity to the sequence GTAAGTATTC.
  • the VP capsid protein encoded by the cap gene can be the capsid protein of any AAV serotype, such as AAV serotype 2, AAV serotype 5, AAV serotype 8, AAV serotype 9, etc.
  • the present invention relates to The intron sequence and splicing strategy are suitable for the correct expression of VP capsid proteins of various existing AAV serotypes.
  • the promoter capable of driving the transcription of cap gene and rep gene in insect cells may be polh promoter or p10 promoter.
  • the cap gene expression cassette uses a p10 promoter
  • the rep gene expression cassette uses a polh promoter.
  • the present invention also provides a nucleic acid molecule comprising the above cap gene expression cassette and/or rep gene expression cassette.
  • the nucleic acid may comprise expression cassettes arranged in tandem, that is, with the same polarity or in an antisense orientation, therefore, the cap gene expression cassette may be in an antisense orientation relative to the rep gene expression cassette, or may be in a sense orientation relative to the rep gene expression cassette.
  • the present invention also provides a recombinant baculovirus vector, preferably an insect cell compatible vector, which comprises a cap gene expression cassette and a rep gene expression cassette, the cap gene expression cassette is preferably the expression cassette constructed in the present invention, the rep gene expression cassette
  • the gene expression cassette constructed in the present invention is preferred.
  • the rAAV vector also contains a foreign gene and AAV inverted terminal repeats located at both ends of the foreign gene, and the ITR acts on the replication, packaging and site-specific integration of AAV, helping the vector to form a stable loop in the host cell. Shaped multimer and chromatin-like structure, so that it can exist stably in the host cell for a long time, and foreign genes can also be continuously and stably expressed.
  • the exogenous gene can be at least one nucleotide sequence encoding a gene of interest (Gene of Interest, GOI) product, and 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, cytokine, interleukin or interferon; it can also be an antigenic protein for the preparation of veterinary or human vaccines, such as hepatitis B surface antigen , rabies virus glycoprotein, foot-and-mouth disease virus capsid protein, etc.; it can also be a reporter protein for evaluating 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 al
  • the present invention also provides the application of the above-mentioned recombinant baculovirus vector in preparation of recombinant adeno-associated virus in insect cells, and introducing the vector into insect cells can realize VP1, VP2, VP3 proteins according to the correct stoichiometric ratio of 1:1 :10 expression, so that packaging produces recombinant adeno-associated virus, its packaging rate is high, and most of the known AAV serotypes (such as AAV2, AAV5, AAV8 and AAV9) have better infectivity; meanwhile, Rep78 and Rep52 proteins The expression level of is controlled within a reasonable range, so that the AAV vector can be stably and mass-produced in insect cells. Also, recombinant baculovirus vectors containing cap gene expression cassettes can be used to prepare AAV capsids in vitro.
  • the present invention also provides an insect cell, which contains the expression cassette of cap gene and/or rep gene of AAV, and may also contain the expression cassette of foreign gene. At least one of these expression cassettes can be integrated into the genome of the host insect cell for stable expression, or the nucleic acid containing these expression cassettes can be carried transiently in the insect cell.
  • the insect cell can be any insect cell, such as but not limited to Spodoptera frugiperda cell, Trichoplusia spp. cell, Drosophila cell or mosquito cell, preferably Spodoptera frugiperda cell sf9.
  • the present invention also provides a cell culture comprising the above-mentioned insect cells and a culture medium.
  • the medium comprises an AAV genome.
  • the present invention also provides a recombinant adeno-associated virus particle, which is obtained by culturing the above insect cells under conditions capable of producing recombinant adeno-associated virus particles, and then recovering them.
  • Example 1 Construction of a recombinant baculovirus vector comprising the AAV serum type 9 cap gene expression cassette
  • the cap gene expression cassette includes the p10 promoter, the artificially constructed sequence, and the encoding AAV serum type 9 that only lacks the translation initiation codon ATG of the VP1 protein from 5' to 3' Nucleotide sequence of VP protein.
  • the nucleotide sequence of the p10 promoter is shown in SEQ ID No.1; the nucleotide sequence of the artificially constructed sequence in the cap gene expression cassette of this embodiment is shown in SEQ ID No.2, which includes two intron splicing site, wherein the nucleotide sequence of the first intron is shown in SEQ ID No.3, and the nucleotide sequence of the second intron is shown in SEQ ID No.4; encoding AAV serum type 9 VP protein
  • the nucleotide sequence of the gene is shown in SEQ ID No.5, and there is at least one nucleotide mutation at its amino terminus, which is used to eliminate possible splicing sites in the coding sequence.
  • the above sequences were connected by artificial direct synthesis or overlap extension PCR amplification to obtain construct 9K-1, the nucleotide sequence of which is shown in SEQ ID No.6.
  • Example 2 Construction of a recombinant baculovirus vector comprising the AAV serotype 2 rep gene expression cassette
  • the rep gene expression cassette includes the polh promoter, the artificially constructed sequence, and the AAV serotype 2 encoding sequence that only lacks the translation initiation codon ATG of the Rep78 protein from 5' to 3' Nucleotide sequence of Rep protein.
  • the nucleotide sequence of the polh promoter is shown in SEQ ID No.7; the nucleotide sequence of the artificially constructed sequence in the rep gene expression cassette of this embodiment is shown in SEQ ID No.2, which includes two intron splicing site, wherein, the nucleotide sequence of the first intron is shown in SEQ ID No.3, and the nucleotide sequence of the second intron is shown in SEQ ID No.4; encoding AAV serum type 2 Rep protein
  • the nucleotide sequence is shown in SEQ ID No.8, and there are multiple nucleotide mutations between the Rep78 translation initiation codon and the Rep52 translation initiation codon, which are used to eliminate the possible presence of Translation initiation site.
  • the above sequences were connected by artificial direct synthesis or overlap extension PCR amplification to obtain construct 2R-1, the nucleotide sequence of which is shown in SEQ ID No.9.
  • cap gene expression cassette This comparative example constructs cap gene expression cassette with reference to the method of Urabe et al.
  • the promoter, cap gene in the expression cassette encodes the AAV serum type 9 VP protein, and the translation initiation codon ATG of VP1 in the coding sequence is mutated to a suboptimal codon ACG.
  • the cap gene expression cassette number is 9K-0, and its nucleotide sequence is shown in SEQ ID No.10.
  • rep gene expression cassette construct rep gene expression cassette with reference to the method of Chinese patent CN103849629A, the promoter of this rep gene expression cassette adopts polh promoter, the rep gene in the expression cassette encodes AAV serum type 2 Rep protein, and The original translation initiation codon ATG of AAV Rep78 protein was replaced with ACG.
  • the rep gene expression cassette number is 2R-0.
  • the recombinant baculovirus vectors Ac1, Ac2 and Ac0 prepared in Example 1, Example 2 and Comparative Example were respectively transfected into host cell lines to obtain recombinant baculoviruses, and VP proteins (VP1, VP2, VP3) and Rep proteins were detected respectively (Rep78, Rep52) expression, the specific operation steps are as follows:
  • the recombinant baculovirus vector DNA was extracted and transfected into Sf9 insect cells to prepare the recombinant baculovirus BEV.
  • the transfected Sf9 insect cells successfully produced BEV, and the further infection of a large number of replicated and proliferated BEVs led to the obvious cytopathic effect (CPE) of Sf9 cells.
  • CPE cytopathic effect
  • the culture supernatant of Sf9 cells with CPE was collected, which contained a large amount of BEV, that is, BEV of passage 0 (P0), and the Sf9 cells containing a large amount of rAAV were collected at the same time.
  • Fig. 4 is a Western Blot detection diagram of VP proteins (VP1, VP2 and VP3) of the recombinant baculovirus vector Ac1 containing the cap gene expression cassette and the control recombinant baculovirus vector Ac0. It can be seen from the figure that the above recombinant baculovirus vectors can all produce VP1, VP2 and VP3 in a relatively appropriate ratio.
  • Fig. 5 is the Western Blot detection chart of the Rep protein (Rep78 and Rep52) of the recombinant baculovirus vector Ac2 comprising the rep gene expression cassette. It can be seen from the figure that the recombinant baculovirus vector Ac2 can produce Rep78 and Rep52 proteins, and the expression level of Rep78 protein is lower than that of Rep52 protein.
  • Example 4 is used to produce the construction of the recombinant AAV bacmid of AAV virus in insect cells
  • the method for constructing a recombinant AAV bacmid for producing AAV virus in insect cells refers to Example 1 in our company's previous patent application CN112553257A, including the following steps:
  • ITR-GOI The ITR core element number is I-G-1, and its nucleotide sequence is shown in SEQ ID No.11.
  • the GOI in the ITR core element uses the red fluorescent protein mcherry gene expression cassette, that is, the expression of mcherry is controlled by the miniEf1a promoter, which is convenient for detecting the activity of rAAV, and the ITR and red fluorescent protein expression cassette are constructed on the shuttle vector pFastDual.
  • the two different recombinant AAV bacmids constructed in this example both contain cap gene expression cassettes, rep gene expression cassettes and ITR core elements, as shown in Table 1.
  • the recombinant AAV bacmids CRI-0 and CRI-1 prepared in Example 4 were respectively transfected into host cell lines to obtain AAV recombinant baculovirus,
  • the above-mentioned recombinant bacmid DNA was extracted and transfected into Sf9 insect cells to prepare recombinant baculoviruses BEV and rAAV.
  • the transfected Sf9 insect cells successfully produced BEV, and the further infection of a large number of replicated and proliferated BEVs led to the obvious cytopathic effect (CPE) of Sf9 cells.
  • CPE cytopathic effect
  • the culture supernatant of Sf9 cells with CPE was collected, which contained a large amount of BEV, that is, BEV of passage 0 (P0), and the Sf9 cells containing a large amount of rAAV were collected at the same time.
  • MOI multiplicity of infection
  • Example 6 The purification of recombinant AAV virus particles and the detection of packaging rate and virus titer
  • Nuclease Benzonase
  • the collected cell lysate and the collected supernatant were PEG-precipitated, resuspended and purified by density gradient centrifugation with iodixanol (see Aslanidi et al., 2009, Proc. NatlAcad. Sci. USA, 206:5059-5064).
  • the final purified finished virus was resuspended with 80 ⁇ L-190 ⁇ L PBS, and 10 ⁇ L of the purified finished virus was run on SDS-PAGE gel for silver staining.
  • Fig. 6 provides the SDS-PAGE silver staining detection chart of the purified recombinant AAV virus particles after the recombinant AAV bacmids CRI-0 and CRI-1 are transfected into the host cells. It can be seen from the results that the recombinant AAV bacmid constructed by the present invention can realize the expression of VP1, VP2 and VP3 proteins in a relatively appropriate ratio in insect cells through the regulation of intron splicing, thereby packaging virions.
  • 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: GGAACCCCTAGTGATGGAGTT and Q-ITR-R: CGGCCTCAGTGAGCGA).
  • the detection of rAAV infection titer was carried out by the detection method in the patent CN112280801A previously applied by our company.
  • For the specific operation method please refer to the method of using plasmid-assisted detection of rAAV infection titer in this patent. The detection results are shown in Table 2.
  • Table 2 The packaging efficiency and infectious titer test results of rAAV virions produced by two different recombinant AAV bacmids
  • insect cells transfected with the recombinant AAV bacmid CRI-1 prepared by the present invention can package recombinant baculovirus, and the packaging rate and virus infection titer are high.
  • the cap gene expression cassette 2K-1 for AAV serotype 2 the cap gene expression cassette 5K-1 for AAV serotype 5, and the cap gene expression cassette 8K-1 for AAV serotype 8 were respectively constructed.
  • the nucleotide sequences of the above-mentioned constructs 2K-1, 5K-1, and 8K-1 are respectively shown in SEQ ID No.12 to SEQ ID No.14.
  • the rep gene expression cassette still uses AAV serotype 2, because the Rep protein of AAV serotype 2 is suitable for the preparation of various serotypes of rAAV.
  • Example 4 in this example, three different recombinant AAV bacmids were constructed, all of which contained cap gene expression cassettes, rep gene expression cassettes and ITR core elements, as shown in Table 3.
  • Example 5 and Example 6 in this example, the above-mentioned recombinant AAV bacmid was transfected into host cells, and the purified recombinant AAV virus particles were subjected to SDS-PAGE silver staining detection, and the experimental results are shown in FIG. 8 .
  • the cell packaging efficiency and virus infection titer of rAAV produced by recombinant AAV bacmids of different serotypes were detected, and the detection results are shown in Table 4.
  • Table 4 Utilizes the packaging rate and infection titer of the rAAV virions produced by 3 different recombinant AAV bacmids in Example 7. Test result table

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Abstract

提供了一种包含重叠开放阅读框的基因的表达盒及其在昆虫细胞中的应用，该表达盒包含从5'至3'的、可操作连接的：能够在昆虫细胞中驱动转录的启动子；人工构建序列；仅缺少第一个翻译起始密码子的重叠开放阅读框；其中，人工构建序列包含在昆虫细胞中有剪接活性的第一内含子和第二内含子，第一内含子和第二内含子共用5'部分或3'部分，人工构建序列中不含翻译起始密码子；在转录后加工过程中，通过第一内含子和第二内含子的选择性剪接作用，能够形成翻译起始密码子AUG。利用内含子选择性剪接作用，调控不同衣壳蛋白以及不同Rep蛋白的相对表达，可在昆虫细胞中大规模生产具有高包装率和感染性的rAAV。

Description

一种包含重叠开放阅读框的基因的表达盒及其在昆虫细胞中的应用 【技术领域】
本发明属于基因工程技术领域，更具体地，涉及一种包含重叠开放阅读框的基因的表达盒及其在昆虫细胞中的应用。
【背景技术】
目前已发现哺乳动物基因组中存在包含部分重叠或完全重叠的开放阅读框(Open Reading Frame,ORF)的基因，而重叠开放阅读框在病毒基因组中尤为常见，包含重叠开放阅读框的重叠基因的表达调控相比非重叠基因复杂很多，往往要借助内含子或多个启动子来进行调控。
腺相关病毒(adeno-associated virus，AAV)，也称腺伴随病毒，属于微小病毒科依赖病毒属，是目前发现的一类结构最简单的单链DNA缺陷型病毒，需要辅助病毒(通常为腺病毒)参与复制。重组腺相关病毒(rAAV)由于具有宿主范围广、免疫原性低、安全性高、可介导外源基因在动物体内长期稳定表达等特点，是目前基因治疗领域最具应用前景的载体之一。随着第一种重组腺相关病毒(rAAV)介导的基因治疗药物的批准，对大规模AAV载体制造技术的需求不断增加(Yla-Herttuala S.,2012,Mol Ther,20:1831-1832)。
腺相关病毒基因组中cap基因编码病毒VP衣壳蛋白，其包含3个重叠的开放阅读框，分别编码VP1、VP2、VP3三种类型的亚基来自野生型病毒的AAV中VP1、VP2和VP3的化学计量比约为1:1:10；rep基因编码Rep78、Rep68、Rep52和Rep40四个重叠的多功能蛋白，主要参与AAV的复制。在哺乳动物细胞中，可依赖内含子两个剪接受体序列的交替使用以及对于VP2次优起始密码子ACG的使用，调控三种AAV衣壳蛋白的正常化学计量比表达，从而包装得到正常的重组腺相关病毒。然而，由于哺乳动物细胞和昆虫细胞中内含子剪接机制的差异，在哺乳动物细胞中出现的表达策略不会在昆虫细胞中复制，从而导致野生型AAV在昆虫细胞中无法包装成合适的衣壳。
为了克服上述问题，Urabe等人开发出了昆虫细胞生产体系，将VP1的起始密码子AUG替换为次优起始密码子ACG，构建一个单一的多顺反子mRNA，该多顺反子mRNA不需要剪接即可表达所有三种AAV2的VP蛋白(Urabe等,2002,Hum Gene Ther,13:1935-1943)。然而AAV的血清型众多且与日俱增，Urabe等人的改造方法并不适用于所有血清型，例如Urabe等人使用ACG作为VP1衣壳蛋白起始密码子的杆状病毒系统中产生的AAV5颗粒具有不佳的感染性。在中国专利CN106544325A提供的方法中使用不同的次优起始密码子CTG加强VP1的表达，尽管这种设计提高了AAV5颗粒的感染性，但这种方法在调节VP1/VP2/VP3相对含量方面缺乏灵活性；同时，该方法似乎也缺乏血清型特异性序列调整所必须的灵活性。在中国专利CN101522903A提供的方法中通过将包含有polh启动子序列的人工内含子插入到VP1的开放阅读框中，利用两个启动子分别表达VP1和VP2/VP3，该设计虽然也能实现VP1/VP2/VP3在同一阅读框中表达，但该方法同样不能有效调节VP1/VP2/VP3相对量，且在不同血清型中VP1/VP2/VP3的相对表达量差异较大，导致生产效率较低。
在AAV昆虫细胞生产体系中，Urabe等人构建了Rep78和Rep52两个独立的表达盒，并插入在同一个杆状病毒载体中。由于Rep78的低表达有利于AAV的包装，Rep52使用了Polh启动子，Rep78则使用了相对Polh启动子启动活性较弱的△IE1启动子(Urabe等,2002,Hum Gene Ther,13:1935-1943)。然而，有研究发现，Urabe等人开发的AAV Rep蛋白表达方法存在杆状病毒载体传代不稳定的问题，为了解决这一问题，中国专利CN103849629A公开了一种昆虫细胞中产生AAV的方法，将AAVRep78蛋白的翻译起始密码子替换成ACG，该方法虽然能实现在同一阅读框的Rep78和Rep52蛋白的表达，但缺乏对Rep78和Rep52蛋白相对表达的调控作用。
因此，开发能够在昆虫细胞中稳定和大规模地生产重组腺相关病毒的手段和方法是非常必要的。
【发明内容】
针对现有技术的缺陷，本发明的目的在于提供一种包含重叠开放阅读框的基因的表达盒及其在昆虫细胞中的应用，能够实现在昆虫细胞中稳定和大规模生产具有较高包装率和感染活性的重组腺相关病毒，且适用于各种血清型。
为实现上述目的，本发明提供了一种用于在昆虫细胞中表达包含重叠开放阅读框的基因的表达盒，其包含从5’至3’的、可操作连接的：
能够在昆虫细胞中驱动转录的启动子；
人工构建序列；
仅缺少第一个翻译起始密码子的重叠开放阅读框；
其中，所述人工构建序列包含在昆虫细胞中有剪接活性的天然的或经过人工改造的第一内含子和第二内含子，所述第一内含子和所述第二内含子共用5’部分或3’部分，所述人工构建序列中不含翻译起始密码子；
在转录后加工过程中，通过所述第一内含子和所述第二内含子的选择性剪接作用，能够在所述人工构建序列中形成翻译起始密码子AUG，从而实现调控所述重叠开放阅读框中不同蛋白编码基因的翻译表达。
优选地，所述第一内含子或所述第二内含子位于ATG中的任意相邻两个核苷酸之间。
优选地，所述人工构建序列包含从5’至3’的、可操作连接的：所述第一内含子的5’部分、腺嘌呤核苷酸(A)、所述第二内含子的5’部分、共用3’部分、胸腺嘧啶核苷酸(T)和鸟嘌呤核苷酸(G)；
或者所述人工构建序列包含从5’至3’的、可操作连接的：所述第一内含子的5’部分、腺嘌呤核苷酸(A)、胸腺嘧啶核苷酸(T)、所述第二内含子的5’部分、共用3’部分和鸟嘌呤核苷酸(G)。
优选地，所述第一内含子和所述第二内含子的5’端核苷酸均为GTNN，所述第一内含子和所述第二内含子的3’端核苷酸均为NNAG，其中N为A、T、C、G四种核苷酸中的任意一种。
优选地，所述包含重叠开放阅读框的基因为AAV的cap基因或AAV的rep基因。
优选地，所述启动子为polh启动子或p10启动子。
优选地，所述包含重叠开放阅读框的基因为AAV的cap基因，所述启动子为p10启 动子。
优选地，所述包含重叠开放阅读框的基因为AAV的rep基因，所述启动子为polh启动子。
按照本发明的另一方面，提供了一种核酸分子，其包含第一表达盒，所述第一表达盒为上述表达盒。
优选地，所述核酸分子还包含第二表达盒，所述第二表达盒为上述表达盒，且所述第二表达盒表达的基因与所述第一表达盒表达的基因不同。
优选地，所述第二表达盒相对于所述第一表达盒为反义方向。
优选地，所述第二表达盒相对于所述第一表达盒为正义方向。
优选地，所述核酸分子还包含外源基因及位于所述外源基因两端的AAV反向末端重复序列。
优选地，所述外源基因为报告基因，所述报告基因为氯霉素乙酰转移酶编码基因、β-半乳糖苷酶编码基因、β-葡萄糖醛酸酶编码基因、海肾荧光素酶编码基因、碱性磷酸酶编码基因、萤火虫荧光素酶编码基因、绿色荧光蛋白编码基因和红色荧光蛋白编码基因中的至少一种。
优选地，所述外源基因为编码药物多肽的基因或编码用于制备兽用或人用疫苗的抗原蛋白的基因。
按照本发明的另一方面，提供了一种载体，其包含上述表达盒。
优选地，所述载体为昆虫细胞相容性载体。
优选地，所述载体为质粒和病毒中的至少一种。
按照本发明的另一方面，提供了上述载体在昆虫细胞中制备重组腺相关病毒的应用。
按照本发明的另一方面，提供了上述载体在体外制备AAV衣壳中的应用，所述包含重叠开放阅读框的基因为AAV的cap基因。
按照本发明的另一方面，提供了一种昆虫细胞，其包含上述表达盒。
优选地，所述表达盒被整合至所述昆虫细胞的基因组中。
优选地，所述昆虫细胞为草地贪夜蛾细胞、粉纹夜蛾细胞、果蝇细胞或蚊子细胞。
按照本发明的另一方面，提供了一种细胞培养物，其包含上述昆虫细胞和培养基。
优选地，所述培养基中包含AAV基因组。
按照本发明的另一方面，提供了一种重组腺相关病毒粒子，其是在能产生重组腺相关病毒粒子的条件下培养上述昆虫细胞，然后回收制得的。
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：
(1)本发明表达盒可用于在昆虫细胞中表达由包含重叠开放阅读框的基因编码的多种多肽，例如重组腺相关病毒的VP蛋白(VP1、VP2、VP3)或Rep蛋白(Rep78、Rep52)，本发明不需要改变野生型AAV中VP1蛋白编码序列或Rep78蛋白编码序列的原始翻译起始密码子ATG，通过在一个启动子下游、起始密码子ATG附近插入两个内含子，利用这两个内含子的剪接作用，对转录后的mRNA进行选择性剪接，获得合适比例的两种或多种mRNA，分别翻译成VP1、VP2/VP3蛋白或Rep78、Rep52蛋白。
(2)本发明利用两个内含子的选择性剪接作用实现对重叠基因表达的调控策略，相 比使用次优密码子的方法，能更有效控制VP1、VP2和VP3蛋白的化学计量比例以及Rep78/Rep52的相对表达，实现在昆虫细胞中稳定和大规模生产重组腺相关病毒。
(3)本发明利用内含子选择性剪接策略，实现在昆虫细胞中生产重组腺相关病毒，并且适用于各种AAV血清型，使得制备rAAV的灵活性更强，应用范围更广。
(4)本发明将AAV的cap基因、rep基因和带有外源基因的ITR核心表达元件构建在同一重组杆状病毒载体中，用该载体转染昆虫宿主细胞，可以稳定、高产量生产重组AAV病毒粒子。
【附图说明】
图1为本发明野生型AAV中cap基因和rep基因表达调控示意图。
图2为本发明实施例1中cap基因表达盒表达调控示意图，其中，内容A为cap基因表达盒DNA链示意图；内容B为在转录后加工过程中第一内含子和第二内含子均未发生剪接时VP2、VP3蛋白翻译表达的示意图；内容C为在转录后加工过程中第二内含子发生剪接后VP1蛋白翻译表达的示意图；内容D为转录后加工过程中第一内含子发生剪接后VP2、VP3蛋白翻译表达的示意图。
图3为本发明实施例2中rep基因表达盒表达调控示意图，其中，内容A为rep基因表达盒DNA链示意图；内容B为在转录后加工过程中第一内含子和第二内含子均未发生剪接时Rep52蛋白翻译表达的示意图；内容C为在转录后加工过程中第二内含子发生剪接后Rep78蛋白翻译表达的示意图；内容D为在转录后加工过程中第一内含子发生剪接后Rep52蛋白翻译表达的示意图。
图4为本发明实施例3中包含有cap基因表达盒的重组杆状病毒载体Ac1以及对比例制备的重组杆状病毒载体Ac0表达VP蛋白的WesternBlot检测图。
图5为本发明实施例3中包含有rep基因表达盒的重组杆状病毒载体Ac2表达Rep蛋白的WesternBlot检测图。
图6为本发明实施例6中重组AAV杆粒CRI-0和CRI-1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，显示了三种衣壳蛋白VP1/VP2/VP3。
图7为本发明实施例6中重组AAV杆粒CRI-0和CRI-1转染宿主细胞后，纯化的重组AAV病毒粒子感染293T细胞的效果图。
图8为本发明实施例7中重组AAV杆粒CRI-2(A)、CRI-3(B)和CRI-4(C)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，显示了三种衣壳蛋白VP1/VP2/VP3。
图9为本发明实施例7中重组AAV杆粒CRI-2、CRI-3和CRI-4转染宿主细胞后，纯化的重组AAV病毒粒子感染293T细胞的效果图。
【具体实施方式】
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。
术语解释
正如本文所用的，术语“可操作连接”是指多核苷酸(或多肽)序列以功能性关系的 连接。当两段核苷酸序列置于功能性关系时，这两段核苷酸序列是“可操作连接”的。例如，转录调控序列(例如启动子)如果影响某基因编码序列的转录，则其与该基因编码序列可操作连接。
术语“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。
术语“开放阅读框”(Open Reading Frame,ORF)是结构基因的正常核苷酸序列，具有编码蛋白质或多肽的潜能，从起始密码子开始，结束于终止密码子，其间不存在使翻译中断的终止密码子。在一条mRNA链上，核糖体从起始密码子开始翻译，沿着mRNA序列合成多肽链并不断延伸，遇到终止密码子时，多肽链的延伸反应终止。
术语“载体”是指设计用于转运、转移和/或存储遗传物质，以及表达遗传物质和/或将遗传物质整合到宿主细胞染色体DNA中的核酸分子，例如质粒载体、粘粒载体、人工染色体、噬菌体载体和其他病毒载体。载体通常由至少三个基本单元组成，即复制源、选择标记和多克隆位点。
术语“内含子”又称间隔顺序，指一个基因或mRNA分子中无编码作用的片段，是真核生物细胞DNA中的间插序列。内含子序列被转录在mRNA前体中，经过剪接被去除，最终不存在于成熟mRNA分子中。根据剪接过程为自发还是要经过剪接体的加工，将内含子分为自剪接内含子和剪接体内含子。自剪接内含子是一种特殊的内含子，是一种核酶，可以通过自身作用被切除，来离开mRNA。本发明涉及的内含子是剪接体内含子，这类内含子的剪除要有剪接体的帮助，内含子序列两端有剪接供体序列和剪接受体序列，是切断和重接位点处两旁的序列。剪接体是由核小RNA(small nuclearRNA，snRNA)和蛋白质因子动态组成的核糖核蛋白复合体，剪接体识别mRNA前体的剪接位点并催化剪接反应，将内含子完全剪除，上下游RNA序列再重新连接。
术语“AAV血清型”：自AAV被发现以来，目前已经从腺病毒、人或灵长类动物和一些哺乳动物分离出超过100种AAV血清型或突变体，其中被应用的主要是AAV1-9。不同血清型的rAAV载体主要区别为衣壳蛋白的不同。不同的AAV血清型在感染效率和组织特异性方面存在一定的差异。
所有已知腺相关病毒血清型的基因组结构非常相似，AAV是单链DNA病毒，基因组结构简单，全长约4.7kb，如图1所示，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)。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AAVDNA，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。Cap基因编码结构性的VP衣壳蛋白，其包含3个重叠的开放阅读框，分别编码VP1、VP2、VP3三种类型的亚基， VP1、VP2和VP3含有不同的起始密码子，共用一个终止密码子，VP1和VP2共享VP3序列。VP1的N端具有一个保守的磷脂酶A2序列，该序列与病毒从体内逃逸有关，并对其感染性至关重要；VP2蛋白对于组装或者感染并非比不可少；VP3蛋白的核心由保守的β-桶基序组成，决定了不同血清型AAV与宿主细胞作用受体的差异。野生型AAV中三种蛋白的正确比例为3:3:54，约为1:1:10。Rep基因编码Rep78、Rep68、Rep52和Rep40四个重叠的多功能蛋白，Rep78和Rep68蛋白参与AAV的复制及整合，可以和ITR中的特定序列结合；Rep52和Rep40蛋白具有解旋酶和ATP酶的活性，在参与单链基因组的复制同时也参与病毒的装配。无论是在哺乳动物细胞中，还是在昆虫细胞中，编码Rep78和Rep52蛋白的未剪接的mRNA足以满足制备rAAV的需求。
本发明提到的核酸分子可以是DNA分子，也可以是RNA分子。本领域技术人员已知，RNA序列与DNA序列基本相似或具有一定程度的序列同一性，DNA序列中的胸腺嘧啶(T)可被认为等同于RNA序列中的尿嘧啶(U)。
本发明提到的内含子可以是天然内含子，也可以是经过人工改造的内含子，且其在昆虫细胞内具有剪接活性，但其来源不局限于昆虫细胞。
本发明提供的一种用于在昆虫细胞中表达包含重叠开放阅读框的基因的表达盒包含从5’至3’的、可操作连接的：
能够在昆虫细胞中驱动转录的启动子；
人工构建序列；
仅缺少第一个翻译起始密码子的重叠开放阅读框；
其中，所述人工构建序列包含在昆虫细胞中有剪接活性的天然的或经过人工改造的第一内含子和第二内含子，所述第一内含子和所述第二内含子共用5’部分或3’部分，所述人工构建序列中不含翻译起始密码子；
在转录后加工过程中，通过所述第一内含子和所述第二内含子的选择性剪接作用，能够在所述人工构建序列中形成翻译起始密码子AUG，从而实现调控所述重叠开放阅读框中不同蛋白编码基因的翻译表达。
一些实施例中，所述第一内含子或所述第二内含子位于ATG中的任意相邻两个核苷酸之间。第一内含子和第二内含子的排布顺序没有特殊限定，以第一内含子位于第二内含子上游为例：当第一内含子位于ATG中的任意相邻两个核苷酸之间、第二内含子中不和第一内含子共用的部分(3’部分)位于该ATG中G的下游时，在转录后加工过程中，通过第一内含子的剪接作用，能够在重叠开放阅读框的上游形成翻译起始密码子AUG；当第二内含子位于ATG中的任意相邻两个核苷酸之间、第一内含子中不和第二内含子共用的部分(5’部分)位于该ATG中A的上游时，在转录后加工过程中，通过第二内含子的剪接作用，能够在重叠开放阅读框的上游形成翻译起始密码子AUG。
一些实施例中，利用内含子选择性剪接作用，包含有两个内含子的表达盒可用于昆虫细胞中AAVVP衣壳蛋白的正确表达或Rep蛋白的优势表达，其中，cap基因中的重叠开放阅读框编码AAV衣壳蛋白VP1、VP2和VP3；rep基因中的重叠开放阅读框编码Rep78和Rep52蛋白。
一些实施例中，利用内含子选择性剪接作用，包含有两个内含子的表达盒可用于昆虫 细胞中表达多种猿猴病毒40(SV40)的VP衣壳蛋白。SV40是具有5.2kb共价闭环的环状基因组的双链DNA病毒，其同样包含三种衣壳蛋白VP1、VP2和VP3，VP3蛋白是VP2蛋白的截短形式，VP3和VP2共用终止密码子，VP1编码序列的5’部分与VP2、VP3编码序列的3’部分重叠，但不具有相同的开放阅读框。在哺乳细胞中，这些VP蛋白的表达受到内含子剪接机制的调控。本发明表达盒用于在昆虫细胞中表达SV40衣壳蛋白时，该表达盒中仅缺少第一个翻译起始密码子的ORF为仅缺少VP2蛋白翻译起始密码子ATG的VP蛋白编码序列，VP2、VP3蛋白的相对表达调控可利用本发明内含子剪接策略实现。
一些实施例中，所述包含重叠开放阅读框的基因为AAV的cap基因，所述仅缺少第一个翻译起始密码子的重叠开放阅读框为仅缺少VP1蛋白翻译起始密码子ATG的VP蛋白编码序列，所述人工构建序列包含从5’至3’的、可操作连接的：所述第一内含子的5’部分、腺嘌呤核苷酸(A)、所述第二内含子的5’部分、共用3’部分、胸腺嘧啶核苷酸(T)和鸟嘌呤核苷酸(G)；或者所述第一内含子的5’部分、腺嘌呤核苷酸、胸腺嘧啶核苷酸、所述第二内含子的5’部分、共用3’部分和鸟嘌呤核苷酸。
本发明对野生型AAVVP1蛋白编码序列自身N端的内含子剪接位点进行突变，使得昆虫细胞中的剪接体无法识别该剪接位点，第一内含子的5’部分和共用3’部分形成完整的第一内含子剪接位点，第二内含子的5’部分和共用3’部分形成完整的第二内含子剪接位点，如图2所示，在转录后加工过程中，若昆虫细胞中的剪接体识别第一内含子剪接位点并催化剪接反应，则在mRNA最前端不能形成完整的AUG-起始位点，核糖体从5’至3’识别VP2蛋白的起始密码子时，VP2蛋白得以翻译表达，由于VP2起始密码子是次优密码子ACG，会造成核糖体扫描泄露，于是VP3蛋白得以翻译表达；若昆虫细胞中的剪接体识别第二内含子剪接位点并催化剪接反应，剪接后形成完整的AUG-起始位点，则mRNA从第一个AUG处翻译，VP1蛋白表达；若昆虫细胞中的剪接体既没有剪接第一内含子剪接位点，又没有剪接第二内含子剪接位点，VP1蛋白编码序列5’端没有起始密码子，VP2、VP3蛋白翻译表达。通过不同内含子不同概率的剪接作用，控制衣壳蛋白VP1、VP2和VP3的相对表达量。
一些实施例中，所述包含重叠开放阅读框的基因为AAV的rep基因，所述仅缺少第一个翻译起始密码子的重叠开放阅读框为仅缺少Rep78蛋白翻译起始密码子ATG的Rep蛋白编码序列，所述人工构建序列包含从5’至3’的、可操作连接的：所述第一内含子的5’部分、腺嘌呤核苷酸、所述第二内含子的5’部分、共用3’部分、胸腺嘧啶核苷酸和鸟嘌呤核苷酸；或者所述第一内含子的5’部分、腺嘌呤核苷酸、胸腺嘧啶核苷酸、所述第二内含子的5’部分、共用3’部分和鸟嘌呤核苷酸。
本发明对野生型AAVRep78蛋白编码序列中Rep52蛋白翻译起始密码子上游的可能的一个或多个翻译起始位点进行突变，使得昆虫细胞中的核糖体无法识别这些位点，第一内含子的5’部分和共用3’部分形成完整的第一内含子剪接位点，第二内含子的5’部分和共用3’部分形成完整的第二内含子剪接位点，如图3所示，在转录后加工过程中，若昆虫细胞中的剪接体识别第一内含子剪接位点并催化剪接反应，则在mRNA最前端不能形成完整的AUG-起始位点，核糖体从5’至3’识别Rep52蛋白的起始密码子时，Rep52蛋白得以翻译表达；若昆虫细胞中的剪接体识别第二内含子剪接位点并催化剪接反应，剪接后形成完整 的AUG-起始位点，则mRNA从第一个AUG处翻译，Rep78蛋白表达；若昆虫细胞中的剪接体既没有剪接第一内含子剪接位点，又没有剪接第二内含子剪接位点，Rep78蛋白编码序列5’端没有起始密码子，Rep52蛋白翻译表达。通过不同内含子不同概率的剪接作用，控制Rep78蛋白和Rep52蛋白的相对表达量。
一些实施例中，优选地，所述第一内含子和所述第二内含子的5’端核苷酸均为GTNN，所述第一内含子和所述第二内含子的3’端核苷酸均为NNAG，其中N代表A、T、C、G四种核苷酸中的任意一种。这样可调控VP1、VP2和VP3蛋白以更接近化学计量比进行翻译表达，有利于包装成合适的衣壳；同样地，使得Rep78蛋白翻译表达量较Rep52蛋白的低，有利于较高的载体产量。
一些实施例中，所述人工构建序列的核苷酸序列为SEQ ID No.2所示的序列。本领域技术人员可知的，其中内含子序列的变化形式可用于本发明技术方案。在某些结构中，可使用与本发明内含子核苷酸序列具有相似性的序列，例如，第一内含子5’部分的5’端具有与序列GTAAGTATTC至少40％或50％或60％或70％或80％或90％同一性的序列，第二内含子5’部分的5’端具有与序列GTAAGTATCG至少40％或50％或60％或70％或80％或90％同一性的序列，第一内含子和第二内含子共用的3’部分的3’端具有与序列AAACATTATTTATTTTGCAG至少40％或50％或60％或70％或80％或90％同一性的序列。
一些实施例中，cap基因编码的VP衣壳蛋白可以是任意AAV血清型的衣壳蛋白，例如AAV血清2型、AAV血清5型、AAV血清8型、AAV血清9型等等，本发明涉及的内含子序列及剪接策略适用于现有的多种AAV血清型的VP衣壳蛋白的正确表达。
一些实施例中，能够在昆虫细胞中驱动cap基因和rep基因转录的启动子可以为polh启动子或p10启动子。优选地，cap基因表达盒采用p10启动子，rep基因表达盒采用polh启动子。
本发明还提供一种核酸分子，包含上述cap基因表达盒和/或rep基因表达盒。核酸可以包含以串联即以相同极性或以反义方向排列的表达盒，因此，cap基因表达盒可以相对于rep基因表达盒为反义方向，也可以相对于rep基因表达盒为正义方向。
本发明还提供一种重组杆状病毒载体，优选为昆虫细胞相容的载体，其包含cap基因表达盒和rep基因表达盒，该cap基因表达盒优选本发明构建的表达盒，rep基因表达盒优选本发明构建的基因表达盒。应当理解的是，该rAAV载体还包含外源基因及位于所述外源基因两端的AAV反向末端重复序列，ITR作用于AAV的复制包装及定点整合，帮助载体在宿主细胞中形成稳定的环状多聚体和类染色质结构，从而能在宿主细胞中长期稳定存在，外源基因也能持续稳定表达。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；也可以是用于制备兽用或人用疫苗的抗原蛋白，例如乙肝表面抗原、狂犬病毒糖蛋白、口蹄疫病毒衣壳蛋白等；还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。
另一方面，本发明还提供上述重组杆状病毒载体在昆虫细胞中制备重组腺相关病毒的应用，将该载体导入昆虫细胞中可以实现VP1、VP2、VP3蛋白按照正确的化学计量比1:1:10表达，从而包装产生重组腺相关病毒，其包装率高，且已知的大部分AAV血清型(例如AAV2、AAV5、AAV8和AAV9)均具有较佳的感染性；同时，Rep78和Rep52蛋白的表达量控制在合理范围内，从而能够在昆虫细胞中稳定、大量生产AAV载体。并且，包含cap基因表达盒的重组杆状病毒载体可用于在体外制备AAV衣壳。
另一方面，本发明还提供一种昆虫细胞，该昆虫细胞包含AAV的cap基因和/或rep基因的表达盒，还可以包含外源基因的表达盒。这些表达盒中的至少一种可整合至宿主昆虫细胞的基因组中稳定表达，也可以是昆虫细胞中瞬时携带含这些表达盒的核酸。
一些实施例中，所述昆虫细胞可以为任意昆虫细胞，例如但不限于草地贪夜蛾细胞(Spodoptera frugiperda cell)、粉纹夜蛾细胞、果蝇细胞或蚊子细胞，优选为草地贪夜蛾细胞sf9。
另一方面，本发明还提供一种细胞培养物，其包含上述昆虫细胞和培养基。
一些实施例中，所述培养基中包含AAV基因组。
另一方面，本发明还提供一种重组腺相关病毒粒子，其是在能产生重组腺相关病毒粒子的条件下培养上述昆虫细胞，然后回收制得的。
以下结合具体实施例，对上述技术方案详细说明。
实施例1构建包含AAV血清9型cap基因表达盒的重组杆状病毒载体
(1)构建cap基因表达盒：参见图2，该cap基因表达盒从5’至3’依次包括p10启动子、人工构建序列和仅缺少VP1蛋白翻译起始密码子ATG的编码AAV血清9型VP蛋白的核苷酸序列。p10启动子的核苷酸序列如SEQ ID No.1所示；本实施例cap基因表达盒中人工构建序列的核苷酸序列如SEQ ID No.2所示，其包含两个内含子剪接位点，其中，第一内含子的核苷酸序列如SEQ ID No.3所示，第二内含子的核苷酸序列如SEQ ID No.4所示；编码AAV血清9型VP蛋白的核苷酸序列如SEQ ID No.5所示，其氨基端存在至少一个核苷酸的突变，用来消除编码序列内可能存在的剪切位点。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9K-1，其核苷酸序列如SEQ ID No.6所示。
(2)构建重组杆状病毒载体：将上述构建物9K-1克隆到pFastBac载体上，制备转移质粒；将上述质粒转化DH10Bac菌株中，通过Tn7转座，获得重组杆状病毒载体Ac1。
实施例2构建包含AAV血清2型rep基因表达盒的重组杆状病毒载体
(1)构建rep基因表达盒：参见图3，该rep基因表达盒从5’至3’依次包括polh启动子、人工构建序列和仅缺少Rep78蛋白翻译起始密码子ATG的编码AAV血清2型Rep蛋白的核苷酸序列。polh启动子的核苷酸序列如SEQ ID No.7所示；本实施例rep基因表达盒中人工构建序列的核苷酸序列如SEQ ID No.2所示，其包含两个内含子剪接位点，其中，第一内含子的核苷酸序列如SEQ ID No.3所示，第二内含子的核苷酸序列如SEQ ID No.4所示；编码AAV血清2型Rep蛋白的核苷酸序列如SEQ ID No.8所示，其在Rep78翻译起始密码子与Rep52翻译起始密码子之间存在多个核苷酸的突变，用来消除这段区域内可能存在的翻译起始位点。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2R-1，其核苷酸序列如SEQ ID No.9所示。
(2)构建重组杆状病毒载体：同实施例1步骤(2)，制备得到含有2R-1的重组杆状病毒载体Ac2。
对比例构建重组杆状病毒载体Ac0及rep基因表达盒2R-0
(1)构建cap基因表达盒：本对比例参照Urabe等人(Urabe等,2002,Hum Gene Ther,13:1935-1943)的方法构建cap基因表达盒，该cap基因表达盒的启动子采用p10启动子，表达盒中的cap基因编码AAV血清9型VP蛋白，并将编码序列中VP1的翻译起始密码子ATG突变成次优密码子ACG。该cap基因表达盒编号为9K-0，其核苷酸序列如SEQ ID No.10所示。
(2)构建重组杆状病毒载体：同实施例1步骤(2)，制备得到含有9K-0的重组杆状病毒载体Ac0。
(3)构建rep基因表达盒：参照中国专利CN103849629A的方法构建rep基因表达盒，该rep基因表达盒的启动子采用polh启动子，表达盒中的rep基因编码AAV血清2型Rep蛋白，并将AAV Rep78蛋白的原始翻译起始密码子ATG替换成ACG。该rep基因表达盒编号为2R-0。
实施例3检测VP蛋白(VP1、VP2、VP3)和Rep蛋白(Rep78、Rep52)表达情况
将实施例1、实施例2及对比例制备的重组杆状病毒载体Ac1、Ac2和Ac0分别转染宿主细胞系培养获得重组杆状病毒，分别检测VP蛋白(VP1、VP2、VP3)和Rep蛋白(Rep78、Rep52)表达情况，具体操作步骤如下：
抽提上述重组杆状病毒载体DNA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(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-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检验VP蛋白(VP1、VP2、VP3)和Rep蛋白(Rep78、Rep52)的表达情况。
图4为包含有cap基因表达盒的重组杆状病毒载体Ac1以及对照重组杆状病毒载体Ac0的VP蛋白(VP1、VP2和VP3)Western Blot检测图。由图看出，以上重组杆状病毒载体都能以相对合适的比例产生VP1、VP2和VP3。
图5为包含有rep基因表达盒的重组杆状病毒载体Ac2的Rep蛋白(Rep78和Rep52)Western Blot检测图。由图看出，重组杆状病毒载体Ac2能够产生Rep78和Rep52蛋白，且Rep78蛋白的表达量低于Rep52蛋白。
实施例4用于在昆虫细胞中生产AAV病毒的重组AAV杆粒的构建
本实施例构建用于在昆虫细胞中生产AAV病毒的重组AAV杆粒的方法参照本公司此前申请专利CN112553257A中的实施例1，包括如下步骤：
(1)构建包含AAV必需功能元件cap和rep基因表达盒的同源重组载体；然后通过Red同源重组在大肠杆菌中将该同源重组载体中的必需功能元件插入到杆状病毒基因组中必需基因Ac135(116492...117391)的C端，获得包含有cap和rep基因表达盒的重组杆状病毒载体，编号为Bac-Cap-Rep。
(2)构建包含ITR核心元件(ITR-GOI)的穿梭载体。ITR核心元件编号为I-G-1，其核苷酸序列如SEQ ID No.11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穿梭载体pFastDual上。
(3)用上述步骤(2)中构建的穿梭载体转化含有Bac-Cap-Rep重组杆粒的感受态细胞，利用Tn7重组将ITR-GOI插入到Bac-Cap-Rep重组杆粒中的Tn7位点上，最终得到包含生产rAAV所必需的功能蛋白组分和ITR核心元件的重组杆状病毒基因组的重组杆粒，编号为Bac-Cap-Rep-ITR-GOI。
本实施例构建的2种不同重组AAV杆粒均包含cap基因表达盒、rep基因表达盒和ITR核心元件，具体如表1所示。
表1实施例4中构建的2种不同重组AAV杆粒的组成元件列表
Figure PCTCN2021135642-appb-000001
实施例5AAV重组杆状病毒的制备
将实施例4中制备的重组AAV杆粒CRI-0和CRI-1分别转染宿主细胞系培养获得AAV重组杆状病毒，
抽提上述重组杆粒DNA转染Sf9昆虫细胞，制备重组杆状病毒BEV和rAAV。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(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-P1，细胞则标记为用BEV-P0包装的rAAV。
实施例6重组AAV病毒粒子的纯化及其包装率、病毒滴度的检测
按照实施例5的操作继续扩大培养，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.NatlAcad.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。
图6提供了重组AAV杆粒CRI-0和CRI-1转染宿主细胞后，纯化的重组AAV病毒粒子进行SDS-PAGE银染的检测图。由结果看出，本发明构建的重组AAV杆粒通过内含子剪接调控作用，能在昆虫细胞中实现VP1、VP2和VP3蛋白以相对合适比例的表达，从而包装出病毒粒子。
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)。rAAV感染滴度的检测采用本公司此前申请专利CN112280801A中的检测方法进行，具体操作方法参见该专利中利用质粒辅助进行rAAV感染滴度检测的方法，检测结果如表2。
表2利用2种不同重组AAV杆粒生产的rAAV病毒粒子的包装率和感染滴度检测结果表
重组AAV杆粒编号 细胞包装率(VG/cell) 病毒感染滴度(VG/mL)
CRI-0 4.75E+05 4.31E+08
CRI-1 7.86E+05 5.37E+08
结合表2和图7可以看出，利用本发明制备的重组AAV杆粒CRI-1转染昆虫细胞能包装出重组杆状病毒，包装率和病毒感染滴度较高。
实施例7不同AAV血清型中内含子调控作用的验证
为了验证以上内含子调控方式能在不同AAV血清型中起作用。参照实施例1中的方法分别构建了AAV血清2型的cap基因表达盒2K-1、AAV血清5型的cap基因表达盒5K-1和AAV血清8型的cap基因表达盒8K-1。上述构建物2K-1、5K-1、和8K-1的核苷酸序列分别如SEQ ID No.12至SEQ ID No.14所示。本实施例rep基因表达盒仍采用的是AAV血清2型，因为AAV血清2型的Rep蛋白适应于各种血清型rAAV的制备。
参照实施例4，本实施例构建了3种不同重组AAV杆粒，它们均包含cap基因表达盒、rep基因表达盒和ITR核心元件，具体如表3所示。
表3实施例7中构建的3种不同重组AAV杆粒的组成元件列表
Figure PCTCN2021135642-appb-000002
参照实施例5和实施例6，本实施例将上述重组AAV杆粒转染宿主细胞，纯化获得的重组AAV病毒粒子进行SDS-PAGE银染检测，实验结果如图8。并对不同血清型的重组AAV杆粒产生的rAAV的细胞包装率和病毒感染滴度进行了检测，检测结果如表4。
表4利用实施例7中3种不同重组AAV杆粒生产的rAAV病毒粒子的包装率和感染滴度 检测结果表
重组AAV杆粒编号 细胞包装率(VG/cell) 病毒感染滴度(VG/mL)
CRI-2 3.59E+05 3.89E+08
CRI-3 3.88E+05 7.46E+08
CRI-4 2.59E+05 9.28E+08
结合表4和图9的结果表明，本发明通过内含子剪接作用调控昆虫细胞中VP1、VP2和VP3蛋白以相对合适比例的表达，适用于各种AAV血清型，均能大规模生产rAAV病毒粒子。
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。

Claims (26)

  1. 一种用于在昆虫细胞中表达包含重叠开放阅读框的基因的表达盒，其特征在于，包含从5’至3’的、可操作连接的：
    能够在昆虫细胞中驱动转录的启动子；
    人工构建序列；
    仅缺少第一个翻译起始密码子的重叠开放阅读框；
    其中，所述人工构建序列包含在昆虫细胞中有剪接活性的天然的或经过人工改造的第一内含子和第二内含子，所述第一内含子和所述第二内含子共用5’部分或3’部分，所述人工构建序列中不含翻译起始密码子；
    在转录后加工过程中，通过所述第一内含子和所述第二内含子的选择性剪接作用，能够在所述人工构建序列中形成翻译起始密码子AUG，从而实现调控所述重叠开放阅读框中不同蛋白编码基因的翻译表达。
  2. 根据权利要求1所述的表达盒，其特征在于：所述第一内含子或所述第二内含子位于ATG中的任意相邻两个核苷酸之间。
  3. 根据权利要求2所述的表达盒，其特征在于，所述人工构建序列包含从5’至3’的、可操作连接的：所述第一内含子的5’部分、腺嘌呤核苷酸、所述第二内含子的5’部分、共用3’部分、胸腺嘧啶核苷酸和鸟嘌呤核苷酸；
    或者所述人工构建序列包含从5’至3’的、可操作连接的：所述第一内含子的5’部分、腺嘌呤核苷酸、胸腺嘧啶核苷酸、所述第二内含子的5’部分、共用3’部分和鸟嘌呤核苷酸。
  4. 根据权利要求1所述的表达盒，其特征在于：所述第一内含子和所述第二内含子的5’端核苷酸均为GTNN，所述第一内含子和所述第二内含子的3’端核苷酸为NNAG，其中N为A、T、C、G四种核苷酸中的任意一种。
  5. 根据权利要求1所述的表达盒，其特征在于：所述包含重叠开放阅读框的基因为AAV的cap基因或AAV的rep基因。
  6. 根据权利要求1所述的表达盒，其特征在于：所述启动子为polh启动子或p10启动子。
  7. 根据权利要求6所述的表达盒，其特征在于：所述包含重叠开放阅读框的基因为AAV的cap基因，所述启动子为p10启动子。
  8. 根据权利要求6所述的表达盒，其特征在于：所述包含重叠开放阅读框的基因为AAV的rep基因，所述启动子为polh启动子。
  9. 一种核酸分子，其特征在于：包含第一表达盒，所述第一表达盒为权利要求1-8任一所述的表达盒。
  10. 根据权利要求9所述的核酸分子，其特征在于：还包含第二表达盒，所述第二表达盒为权利要求1-8任一所述的表达盒，且所述第二表达盒表达的基因与所述第一表达盒表达的基因不同。
  11. 根据权利要求10所述的核酸分子，其特征在于：所述第二表达盒相对于所述第一表达盒为反义方向。
  12. 根据权利要求10所述的核酸分子，其特征在于：所述第二表达盒相对于所述第一表达盒为正义方向。
  13. 根据权利要求9所述的核酸分子，其特征在于：还包含外源基因及位于所述外源基因两端的AAV反向末端重复序列。
  14. 根据权利要求13所述的核酸分子，其特征在于：所述外源基因为报告基因，所述报告基因为氯霉素乙酰转移酶编码基因、β-半乳糖苷酶编码基因、β-葡萄糖醛酸酶编码基因、海肾荧光素酶编码基因、碱性磷酸酶编码基因、萤火虫荧光素酶编码基因、绿色荧光蛋白编码基因和红色荧光蛋白编码基因中的至少一种。
  15. 根据权利要求13所述的核酸分子，其特征在于：所述外源基因为编码药物多肽的基因或编码用于制备兽用或人用疫苗的抗原蛋白的基因。
  16. 一种载体，其特征在于：包含权利要求1-8任一所述的表达盒。
  17. 根据权利要求16所述的载体，其特征在于：所述载体为昆虫细胞相容性载体。
  18. 根据权利要求16所述的载体，其特征在于：所述载体为质粒和病毒中的至少一种。
  19. 权利要求16所述的载体在昆虫细胞中制备重组腺相关病毒的应用。
  20. 权利要求16所述的载体在体外制备AAV衣壳中的应用，其特征在于：所述包含重叠开放阅读框的基因为AAV的cap基因。
  21. 一种昆虫细胞，其特征在于：包含权利要求1-8任一所述的表达盒。
  22. 根据权利要求21所述的昆虫细胞，其特征在于：所述表达盒被整合至所述昆虫细胞的基因组中。
  23. 根据权利要求21所述的昆虫细胞，其特征在于：所述昆虫细胞为草地贪夜蛾细胞、粉纹夜蛾细胞、果蝇细胞或蚊子细胞。
  24. 一种细胞培养物，其特征在于：包含权利要求21所述的昆虫细胞和培养基。
  25. 根据权利要求24所述的细胞培养物，其特征在于：所述培养基中包含AAV基因组。
  26. 一种重组腺相关病毒粒子，其特征在于：是在能产生重组腺相关病毒粒子的条件下培养权利要求21所述的昆虫细胞，然后回收制得的。
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