Abrégé

A baculovirus vector and a use thereof in the preparation of a recombinant adeno-associated virus (rAAV) in an insect cell are provided. The baculovirus vector includes an exogenous gene expression cassette and a stable sequence. The stable sequence is located at a site 5 kb or less from the exogenous gene expression cassette, and the stable sequence is a conserved noncoding element (CNE) sequence or a nucleocapsid assembly-essential element (NAE) sequence. When an insect cell is infected with a recombinant baculovirus (rBV) constructed in this way, after multiple continuous passages, production levels of the rBV and the rAAV still remain relatively stable.

Classes IPC  ?

• C12N 15/86 - Vecteurs viraux
• C12N 5/07 - Cellules animales ou tissus animaux
• C12N 7/00 - Virus, p. ex. bactériophagesCompositions les contenantLeur préparation ou purification

Abrégé

An expression cassette containing overlapping open reading frames and an application thereof are provided. The overlapping open reading frames are overlapping open reading frames of a first ORF and a second ORF and include in sequence from a 5′ end to a 3′ end: a first promoter at least used to drive gene transcription of the first ORF; a 5′ part of a gene of the first ORF; an intron; and a 3′ part of a gene of the second ORF, the intron including a second promoter used only to drive gene transcription of the second ORF. By arranging two promoters in a single expression cassette in the disclosure, the two promoters are used to drive the expression of proteins of the overlapping reading frames and regulate the relative expression time and expression intensity of different proteins.

Classes IPC  ?

• C12N 15/86 - Vecteurs viraux
• C12N 5/07 - Cellules animales ou tissus animaux

Abrégé

Disclosed in the present invention are an expression cassette containing overlapping open reading frames (ORFs), and a use. The overlapping ORFs are overlapping ORFs comprising a first ORF and a second ORF, and the expression cassette sequentially comprises from a 5' end to a 3' end: at least a first promoter used for driving transcription of a gene of the first ORF; a 5' part of the gene of the first ORF; an intron; and a 3'part of a gene of the second ORF, the intron comprising a second promoter only used for driving transcription of the gene of the second ORF. According to the present invention, two promoters are provided in a single expression cassette and are respectively used for driving expression of proteins of overlapping reading frames and adjusting the relative expression time and expression intensity of different proteins, thereby improving the production efficiency of rAAV vectors in insect cells, and also improving the quality of produced rAAV.

Classes IPC  ?

• C12N 15/866 - Vecteurs baculoviraux
• C12N 15/864 - Vecteurs parvoviraux
• C12N 5/10 - Cellules modifiées par l'introduction de matériel génétique étranger, p. ex. cellules transformées par des virus
• C12N 7/01 - Virus, p. ex. bactériophages, modifiés par l'introduction de matériel génétique étranger
• C12R 1/93 - Virus des animaux

Abrégé

The disclosure discloses an expression cassette for expressing a gene including overlapping open reading frames in an insect cell and an application thereof. The expression cassette includes from 5′ to 3′ and operably linked: a promoter capable of driving transcription in the insect cell; an artificially constructed sequence; the overlapping open reading frames missing only a first translation start codon; wherein the artificially constructed sequence includes a native or engineered intron with splicing activity in the insect cell, the intron includes ATG or the intron is located between any two adjacent nucleotides in ATG. A recombinant adeno-associated virus vector including the expression cassette of the disclosure regulates relative expressions of VP1, VP2, and VP3 proteins, and relative expressions of Rep78 and Rep52 proteins by using a designed intron sequence and through an intron splicing function for large-scale production of rAAV.

Classes IPC  ?

• C12N 15/86 - Vecteurs viraux
• C12N 7/00 - Virus, p. ex. bactériophagesCompositions les contenantLeur préparation ou purification

Abrégé

Provided are a nucleic acid for producing rAAV in an insect cell, a VP1 capsid protein mutant, and use. The nucleic acid comprises nucleotide sequences encoding adeno-associated viruses VP1, VP2, and VP3 proteins. Compared with the capsid protein of wild-type AAV serotype 8, the amino acid sequence of the VP1 protein comprises a mutation at one or more sites among sites 84, 92, and 105, and the amino acid sequence of the VP2 protein is not mutated. The VP1 protein coding sequence is mutated, so that degradation of the VP1 protein in an insect cell can be reduced, and the expressed VP1, VP2, and VP3 can maintain a proper ratio to correctly assemble a capsid, thereby improving the yield and effectiveness of producing rAAV by using a baculovirus/insect cell expression system.

Classes IPC  ?

• C12N 15/35 - Parvoviridae, p. ex. virus de l'aleucémie féline, parvovirus humain
• C12N 15/866 - Vecteurs baculoviraux
• C12N 15/65 - Introduction de matériel génétique étranger utilisant des vecteursVecteurs Utilisation d'hôtes pour ceux-ciRégulation de l'expression utilisant des marqueurs
• C12N 5/10 - Cellules modifiées par l'introduction de matériel génétique étranger, p. ex. cellules transformées par des virus
• C12N 7/01 - Virus, p. ex. bactériophages, modifiés par l'introduction de matériel génétique étranger
• C07K 14/015 - Parvoviridae, p. ex. virus de l'aleucémie féline, parvovirus humain

Abrégé

The disclosure belongs to the field of genetic engineering and discloses an expression cassette of a gene including overlapping open reading frames and an application thereof in an insect cell. The expression cassette includes from 5′ to 3′ and operably linked: a promoter capable of driving transcription in the insect cell; an artificially constructed sequence; overlapping open reading frames missing only a first translation start codon; wherein the artificially constructed sequence includes a first intron and a second intron with splicing activity in the insect cell, the first intron and the second intron share a 5′ part or a 3′ part, and the artificially constructed sequence does not include a translation start codon; during a post-transcriptional processing process, through an alternative splicing function of the first intron and the second intron, a translation start codon AUG can be formed.

Classes IPC  ?

• C12N 15/86 - Vecteurs viraux
• C12N 7/00 - Virus, p. ex. bactériophagesCompositions les contenantLeur préparation ou purification
• C07K 14/005 - Peptides ayant plus de 20 amino-acidesGastrinesSomatostatinesMélanotropinesLeurs dérivés provenant de virus

Abrégé

Provided are an expression cassette of a gene containing an overlapped open reading frame and an application of the expression cassette in insect cells. The expression cassette comprises: a promoter capable of driving transcription in the insect cells, an artificially constructed sequence, and an overlapped open reading frame only lacking a first translation initiation codon which are operatively connected from 5'- to 3'- terminal, wherein the artificially constructed sequence contains a first intron and a second intron which have splicing activity in the insect cells, the first intron and the second intron share a 5' part or a 3' part, and the artificially constructed sequence does not contain a translation initiation codon; and in a post-transcription processing process, a translation initiation codon AUG can be formed by means of the alternative splicing effect of the first intron and the second intron. The relative expression of different capsid proteins and different Rep proteins is regulated and controlled by using the alternative splicing effect of the introns, and rAAV with high packaging rate and infectivity can be produced in the insect cells on a large scale.

Classes IPC  ?

• C12N 15/866 - Vecteurs baculoviraux
• C12N 5/10 - Cellules modifiées par l'introduction de matériel génétique étranger, p. ex. cellules transformées par des virus
• C12N 15/35 - Parvoviridae, p. ex. virus de l'aleucémie féline, parvovirus humain
• C07K 14/015 - Parvoviridae, p. ex. virus de l'aleucémie féline, parvovirus humain

Abrégé

Provided are a preparation method and system for a recombinant adeno-associated virus (rAAV) and a recombinant bacmid. The method comprises: first reconstructing a recombinant bacmid containing a recombinant baculovirus genome that produces essential functional elements for an rAAV, at least one of the essential functional elements being inserted into the N-terminal or C-terminal of a locus of an essential gene of the recombinant baculovirus genome; and then transfecting the obtained recombinant bacmid containing the recombinant baculovirus genome that produces the rAAV into a host cell line for culturing to prepare an rAAV. Compared with recombinant baculoviruses obtained by conventional Tn7 recombinant preparations of recombinant bacmid, the recombinant baculovirus obtained by inserting a core element containing Cap, Rep and ITR into two sides of a baculovirus essential gene has a more stable rAAV serial passage production level in a cell and has a higher rAAV yield.

Classes IPC  ?

• C12N 15/86 - Vecteurs viraux

Abrégé

Provided are an expression cassette for expressing a gene containing an overlapping open reading frame in an insect cell, and the use thereof. The expression cassette contains, from 5' to 3', a promoter capable of driving transcription in an insect cell, an artificial construction sequence, and an overlapping open reading frame only lacking a first translation start codon, which are operably linked, wherein the artificial construction sequence contains a natural or artificially modified intron having a splicing activity in the insect cell, and the intron contains ATG, or the intron is located between any two adjacent nucleotides in ATG. Using a designed intron sequence, a recombinant adeno-associated virus vector containing the expression cassette regulates the relative expression of VP1, VP2 and VP3 proteins and the relative expression of Rep78 and Rep52 proteins by means of the splicing effect of the intron, and can mass produce rAAV with a high packaging rate and infectivity in an insect cell.

Classes IPC  ?

• C12N 15/866 - Vecteurs baculoviraux
• C12N 7/01 - Virus, p. ex. bactériophages, modifiés par l'introduction de matériel génétique étranger
• C12N 5/10 - Cellules modifiées par l'introduction de matériel génétique étranger, p. ex. cellules transformées par des virus
• C12R 1/93 - Virus des animaux