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Baculovirus vector and application thereof in preparation of rAAV in insect cells

A baculovirus vector, insect cell technology, applied in the field of genetic engineering, can solve the problems of unstable passage and achieve the effect of stable production level

Pending Publication Date: 2022-07-12
睿征医药科技(武汉)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the defects of the prior art, the purpose of the present invention is to provide a baculovirus vector and its application in preparation of rAAV in insect cells, aiming to solve the problem of unstable passage in the preparation of rAAV by recombinant baculovirus

Method used

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  • Baculovirus vector and application thereof in preparation of rAAV in insect cells
  • Baculovirus vector and application thereof in preparation of rAAV in insect cells
  • Baculovirus vector and application thereof in preparation of rAAV in insect cells

Examples

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Embodiment 1

[0081] Example 1 Construction of bacmid ΔCNE-Bac lacking CNE sequence and bacmid ΔNAE-Bac red lacking NAE sequence Recombination is a highly efficient recombination method at the bacterial level, which can be used for rapid recombination in Escherichia coli (DH10Bac) Baculovirus genome. Red recombination is the use of λ phage Red recombinase (composed of three proteins, Exo, Beta and Gam) to carry out homologous recombination between the linear DNA fragment carrying the homology arm and the specific target sequence of the genome, so as to achieve the target gene recombination. Substitution (Doublet et al., 2008, J Microbiol Methods, 75(2):359-361).

[0082] First construct the first homologous recombination expression box (SEQ ID No. 1) targeting CNE sequence, as figure 1 As shown, the expression cassette includes CNE upstream homologous sequence, chloramphenicol (Chol) resistance gene expression cassette and CNE downstream homologous sequence sequentially from 5' to 3'; The...

Embodiment 2

[0084]Example 2 Construction of recombinant transfer vectors pFastBac-Rep-Cap9, pFastBac-Rep-CNE-Cap9, pFastBac-ITR and pFastBac-ITR-CNE without or with CNE sequences

[0085] A recombinant transfer vector containing AAV Cap expression cassette and Rep expression cassette was constructed. First, the Cap gene sequence of AAV9 and the Rep gene sequence of AAV2 were obtained by PCR amplification from the AAV genome, and then the Cap gene sequence (SEQ ID No. ) into the pFastBac vector to obtain the recombinant transfer vector pFastBac-Rep-Cap9, such as image 3 shown.

[0086] A recombinant transfer vector containing CNE sequence, AAV Cap expression cassette and Rep expression cassette was constructed. First, the CNE sequence (SEQ ID No. 5) was obtained by PCR amplification from the baculovirus genome, and then the CNE sequence was inserted into the pFastBac-Rep-Cap9 vector by restriction enzyme ligation to obtain the recombinant transfer vector pFastBac-Rep-CNE- Cap9, such as...

Embodiment 3

[0089] Example 3 Construction of recombinant transfer vectors pFastBac-Rep-NAE-Cap9 and pFastBac-ITR-NAE containing NAE sequences

[0090] A recombinant transfer vector containing NAE sequence, AAV Cap expression cassette and Rep expression cassette was constructed. First, the NAE sequence (SEQ ID No. 7) was obtained by PCR amplification from the baculovirus genome, and then the NAE sequence was inserted into the pFastBac-Rep-Cap9 vector constructed in Example 2 by restriction enzyme ligation to obtain a recombinant transfer vector pFastBac-Rep-NAE-Cap9, as in Figure 7 shown.

[0091] A transfer vector containing NAE sequence and ITR-GOI was constructed. First, the NAE sequence was obtained by PCR amplification from the baculovirus genome, and then the NAE sequence was inserted into the pFastBac-ITR vector constructed in Example 2 by enzyme ligation to obtain the recombinant transfer vector pFastBac-ITR-NAE, such as Figure 8 shown.

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Abstract

The invention belongs to the technical field of gene engineering, and particularly discloses a baculovirus vector and application of the baculovirus vector in preparation of rAAV in insect cells, the baculovirus vector comprises an exogenous gene expression cassette and a stable sequence, the stable sequence is located within 5kb from the exogenous gene expression cassette, and the stable sequence is a CNE sequence or an NAE sequence. According to the invention, a CNE (or NAE) sequence is constructed near an exogenous gene expression cassette on a recombinant baculovirus vector, so that the recombinant baculovirus carrying an exogenous gene expression cassette such as a functional protein expression cassette or an ITR core expression element and the like necessary for producing AAV infects insect cells; and the production levels of the recombinant baculovirus and the rAAV can still be kept relatively stable.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and more particularly, relates to a baculovirus vector and its application in preparing rAAV in insect cells. Background technique [0002] Recombinant adeno-associated virus (rAAV) is one of the most promising vectors in the field of gene therapy. At present, there are two main types of rAAV production systems: one is a conventional production system using mammalian cells (such as 293 cells, COS cells, HeLa cells, KB cells, etc.); the other is a production system using insect cells. In mammalian cell production systems, the yield of rAAV particles from a single cell is low, and there is a high risk of contamination in culture, which limits the large-scale production and application of rAAV in mammalian cells. [0003] In order to overcome the above problems, a production system for rAAV production by infecting insect cells with baculovirus has been developed. The rep gene, cap gene ...

Claims

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Application Information

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IPC IPC(8): C12N15/866C12N7/00C12N5/10C12N15/34C12R1/93
CPCC12N15/86C12N7/00C12N5/0601C07K14/005C12N2710/14021C12N2710/14043C12N2710/14052C12N2800/105C12N2750/14121C12N2750/14152C12N2750/14122C12N2510/02Y02A50/30
Inventor 不公告发明人
Owner 睿征医药科技(武汉)有限公司
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