CRICPR-Cas9-based method for in-vitro modifying adenovirus vectors

A carrier and gene technology, applied in the field of in vitro modification of type 5 adenovirus ciliary gene based on CRICPR-Cas9, can solve problems such as secondary mutation and restriction enzyme site modification

Active Publication Date: 2016-05-25
BEIJING PROTEOME RES CENT
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Problems solved by technology

[0005] In order to solve the problem that it is difficult to find suitable restriction enzyme sites for transformation of large plasmid vectors with conventional molecular biology methods, and point mutation kits are likely to cause secondary mu...

Method used

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  • CRICPR-Cas9-based method for in-vitro modifying adenovirus vectors
  • CRICPR-Cas9-based method for in-vitro modifying adenovirus vectors
  • CRICPR-Cas9-based method for in-vitro modifying adenovirus vectors

Examples

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Embodiment 1

[0066] Example 1, construction of Ad5F35 chimeric adenovirus vector

[0067] This example takes the construction of the Ad5F35 chimeric adenovirus vector as an example to specifically illustrate that in order to solve the problem that it is difficult to find suitable restriction enzyme sites for transformation of large plasmid vectors using conventional molecular biology methods, the present invention A specific transformation method for large plasmid vectors is provided. This embodiment provides corresponding technical solutions, based on CRICPR-Cas9 technology (Cas9 in vitro realizes site-specific cleavage break site schematic diagram as shown in figure 1 shown) to replace the coding genes of the ciliated head section and rod region of type 5 adenovirus with those of type 35 adenovirus type 35, so as to obtain the Ad5F35 chimeric adenovirus vector. Schematic diagram of the Ad5 cilia gene replacement strategy on the pAD / PL-DEST vector. figure 2 shown.

[0068] 1. Design...

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Abstract

The invention discloses a CRICPR-Cas9-based method for in-vitro modifying adenovirus vectors and further provides a molecular cloning method suitable for modifying large plasmid vectors. The CRICPR-Cas9-based method includes: designing proper sgRNA according to a to-be-modified site of a starting plasmid; adopting Cas9 protein and the sgRNA to in-vitro cut the starting plasmid; connecting the starting plasmid which is cut with a target gene on the basis of a homologous recombination method to complete modifying. By using the CRICPR-Cas9-based method, the problem that proper restriction enzyme cutting sites are difficult to find for modifying the large plasmid vectors through conventional molecular biologic means is solved effectively. The method is an improvement of a site mutation kit which generally includes site mutation of target protein and building of truncation with deleted structural domain. By the method, the technical problem that secondary mutation (random mutation caused by PCR enzyme fidelity) is easy to be caused when the site mutation kit is used for mutation of large vectors exceeding 10kb is solved effectively.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a method for modifying an adenovirus vector in vitro based on CRICPR-Cas9, in particular to a method for modifying type 5 adenovirus ciliary genes in vitro based on CRICPR-Cas9. Background technique [0002] Adenovirus type 5 (Adenovirus type 5, Ad5) is currently the main delivery tool used in the preclinical treatment and clinical treatment research of various major diseases. The cilium of adenovirus consists of three parts: tail, rod, and head section, which play an important role in the process of infecting cells. The process of adenovirus infection of cells begins with the binding of the head section of the cilia to specific receptors on the cell surface. Different serotypes There are large differences in the cell surface receptors recognized by adenovirus (NicklinSA, WuE, NemerowGR, et al. The influence of adenovirus fiber structure and function on vector development for gen...

Claims

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Application Information

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IPC IPC(8): C12N15/66
CPCC07K14/005C12N15/66C12N2710/10022C12N2710/10041C12N2800/80C12N2810/10
Inventor 张普民唐立春张卜兮
Owner BEIJING PROTEOME RES CENT
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