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Expression cassette for expressing genes containing overlapped open reading frames in insect cells and application thereof

An open reading frame, insect cell technology, applied in the field of genetic engineering, can solve the problems of unstable passage of baculovirus vector and lack of regulatory effect

Active Publication Date: 2022-01-07
JINFAN BIOMEDICAL TECH (WUHAN) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies have found that the AAV Rep protein expression method developed by Urabe et al. has the problem of unstable passage of baculovirus vectors. In order to solve this problem, Chinese patent CN 103849629 A discloses a method for producing AAV in insect cells. Replace the translation start codon of AAV Rep78 protein with ACG, although this method can realize the expression of Rep78 and Rep52 proteins in the same reading frame, but it lacks the regulation effect on the relative expression of Rep78 and Rep52 proteins

Method used

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  • Expression cassette for expressing genes containing overlapped open reading frames in insect cells and application thereof
  • Expression cassette for expressing genes containing overlapped open reading frames in insect cells and application thereof
  • Expression cassette for expressing genes containing overlapped open reading frames in insect cells and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0137] Example 1 Construction of a recombinant baculovirus vector comprising AAV serum type 9 cap gene expression cassette I

[0138] (1) Construction of cap gene expression cassette I: see figure 2 , the cap gene expression cassette I includes a p10 promoter, an intron, a nucleotide sequence encoding a T2A peptide and an encoding AAV serum type 9 VP protein that only lacks the translation initiation codon ATG of the VP1 protein from 5' to 3' in sequence the nucleotide sequence. The nucleotide sequence of the p10 promoter is shown in SEQ ID No.1; in the cap gene expression box I, this embodiment specifically provides 4 kinds of introns, and its nucleotide sequences are as shown in SEQ ID No.2 to As shown in SEQ ID No.5, these introns all include the translation initiation codon ATG; the nucleotide sequence encoding T2A peptide is shown in SEQ ID No.6; the nucleotide encoding AAV serum type 9 VP protein The sequence is shown as SEQ ID No.7, and there is at least one nucleoti...

Embodiment 2

[0140] Example 2 Construction of a recombinant baculovirus vector comprising AAV serotype 2 rep gene expression cassette I

[0141] (1) Construction of rep gene expression cassette I: see Figure 5 , the rep gene expression cassette I includes polh promoter, intron, nucleotide sequence encoding T2A peptide and the encoding AAV serum type 2 Rep protein that only lacks the translation initiation codon ATG of Rep78 protein from 5' to 3' the nucleotide sequence. The nucleotide sequence of the polh promoter is shown in SEQ ID No.12; The nucleotide sequence of the intron in the rep gene expression cassette of the present embodiment is shown in SEQ ID No.5, and the intron includes the translation initiation Initiation codon ATG; The nucleotide sequence encoding T2A peptide is shown in SEQ ID No.6; The nucleotide sequence encoding AAV serum type 2 Rep protein is shown in SEQ ID No.13, which is translated at the Rep78 initiation codon There are multiple nucleotide mutations between t...

Embodiment 3

[0143] Example 3 Construction of a recombinant baculovirus vector comprising AAV serotype 9 cap gene expression cassette II

[0144] (1) Construction of cap gene expression cassette II: see image 3 , the cap gene expression cassette II includes the p10 promoter, the artificial construction sequence and the nucleotide sequence encoding the AAV serum type 9 VP protein that only lacks the translation initiation codon ATG of the VP1 protein from 5' to 3'. The nucleotide sequence of the p10 promoter is shown in SEQ ID No.1; in the cap gene expression box II, this embodiment specifically provides two kinds of artificially constructed sequences, the nucleotide sequences of which are respectively shown in SEQ ID No.15 and As shown in SEQ ID No.16, these artificially constructed sequences all include the translation initiation codon ATG, and include two intron splice sites; the nucleotide sequence encoding AAV serum type 9 VP protein is shown in SEQ ID No. As shown in 7, there is at ...

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Abstract

The invention belongs to the technical field of gene engineering, and discloses an expression cassette for expressing a gene containing an overlapped open reading frame in insect cells and application thereof, the expression cassette comprises a promoter operably linked from 5' to 3' for driving transcription in the insect cells; an artificially constructed sequence; and an overlapping open reading frame only lacking a first translation initiation codon; wherein the artificially constructed sequence comprises a natural or artificially modified intron with splicing activity in an insect cell, and the intron comprises ATG, or the intron is positioned between any two adjacent nucleotides in the ATG. The recombinant adeno-associated virus vector containing the expression cassette utilizes a designed intron sequence, regulates the relative expression of VP1, VP2 and VP3 proteins and the relative expression of Rep78 and Rep52 proteins through the splicing effect of intron, and can produce rAAV with high packaging rate and infectivity in insect cells on a large scale.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and more specifically relates to an expression cassette for expressing genes containing overlapping open reading frames in insect cells and an application thereof. Background technique [0002] Adeno-associated virus (adeno-associated virus, AAV), also known as adeno-associated virus, belongs to the genus of Parvoviridae and is the simplest single-stranded DNA-deficient 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. . With the approval of the first recombinant adeno-associated virus (rAAV)-mediated gene therapy drug, there is an increasing demand for large-scale AAV vector manufacturing technology (Yla-Herttuala S., 2012, Mol Ther, 2...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N7/01C12N5/10C12R1/93
CPCC12N15/86C12N7/00C12N5/0601C12N2830/42C12N2800/105C12N2840/50C12N2510/02C12N2710/14043C12N2750/14152Y02A40/146C12N2710/14143C12N2750/14143
Inventor 不公告发明人
Owner JINFAN BIOMEDICAL TECH (WUHAN) CO LTD