Site-directed modification method for DNA viral genome

A DNA virus and fixed-point transformation technology, which is applied in plant gene improvement, recombinant DNA technology, genetic engineering, etc., can solve the problems of low recombination rate, complicated operation of inserting foreign fragments, and difficult point-directed mutation of DNA virus genome, so as to achieve improved transformation The effect of improving efficiency and mutation rate

Active Publication Date: 2013-11-20
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the difficulty in inducing site-directed mutation of DNA virus genomes in the prior art, complicated operation of inserting exogenous fragments, and low recombination rate, the present invention provides a method for site-directed transformation of DNA virus genomes, which can efficiently carry out site-directed transformation of DNA virus genomes, Realize site-directed mutation and specific gene knockout, site-directed insertion of foreign genes, large fragment deletion of specific sequences and site-directed homologous recombination

Method used

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  • Site-directed modification method for DNA viral genome
  • Site-directed modification method for DNA viral genome
  • Site-directed modification method for DNA viral genome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Targeted Knockout of Herpes Simplex Virus Genome TK Gene

[0045] Use the strain of herpes simplex virus type I (HSV1), which can be isolated and preserved by existing methods, for example, infect Vero cells with the fluid from a patient with clinical herpes simplex type I, and amplify to obtain HSV1 8F virus , wherein, 8F is the code name of the virus strain in the preparation process.

[0046] The site-directed knockout transformation method of the herpes simplex virus genome TK gene is realized through the following steps:

[0047] Step 1: After digesting and purifying the AG230 plasmid with BbsI, use T4 DNA ligase (NEB) to connect into the DNA via P109 (5'- CAC CGA GGG CGC AAC GCC GTA CGT -3') and P110 (5'- AAA CAC GTA CGG CGT TGC GCC CTC -3') The double-stranded insert sequence formed by the annealing of two synthetic primers was used to construct plasmid pCW206, and the plasmid pCW206 was extracted by an endotoxin-free plasmid extraction kit for use; ...

Embodiment 2

[0056] Example 2: Rapid site-specific insertion of exogenous red fluorescent protein into recombinant adenovirus vector

[0057]The recombinant adenovirus ADV-152 expressing green fluorescent protein was used. This strain used stratagene’s AdEasy? After infecting AD293 cells, ADV-152 recombinant adenovirus can be prepared and amplified. In this example, green fluorescent protein is replaced with red fluorescent protein. Among them, 152 in ADV-152 is the code name of the virus strain in the preparation process.

[0058] The transformation method of point-directed insertion of exogenous red fluorescent protein of recombinant adenovirus vector is realized through the following steps:

[0059] Step 1: After digesting and purifying the AG230 plasmid with BbsI, use T4 DNA ligase (NEB) to connect to the DNA via P069 (5'- CAC CGC TGA AGC ACT GCA CGC CGT -3') and P070 (5'- AAA CAC GGC GTG CAG TGC TTC AGC -3') The double-stranded insert sequence formed by the annealing of two synthet...

Embodiment 3

[0066] Example 3: Large fragment deletion of specific sequence of recombinant adenovirus

[0067] The recombinant adenovirus ADV-152 expressing green fluorescent protein was used. This strain used stratagene’s AdEasy? After infecting AD293 cells, the virus ADV-152 can be prepared and amplified.

[0068] The large-segment deletion transformation method of the specific sequence of the recombinant adenovirus genome is realized through the following steps:

[0069] Step 1: After digesting and purifying the AG230 plasmid with BbsI, use T4 DNA ligase (NEB) to connect into the DNA via P067 (5'- CAC CGG AGC GCA CCA TCT TCT TCA -3') and P068 (5'- AAA CTG AAG AAG ATG GTG CGC TCC -3') The double-stranded insert sequence formed by the annealing of two synthetic primers was used to construct the plasmid pCW174; then the AG230 plasmid was digested and purified with BbsI, and then ligated into the pCW174 via T4 DNA ligase (NEB) (5'- CAC CGC TGA AGC ACT GCA CGC CGT -3') and P070 (5'- AAA CA...

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Abstract

The invention provides a site-directed modification method for DNA viral genome, and the problems in the prior art are solved that induction of site-directed mutagenesis of DNA viral genome is difficult, the operation of inserting an exogenous fragment is complex, and recombination rate is lower. The site-directed modification method comprises: transfecting cells by a plasmid carrying a nuclease system, infecting by a virus, after the cells show pathological changes, collecting the cells with pathological changes, performing freeze-thaw or ultrasonic processing, and centrifuging, separating the liquid supernatant to obtain a progeny virus. The site-directed modification method is capable of realizing applications to screening of virus attenuated vaccine strains, construction of viral genetic carriers and an oncolytic virus, research on virus function sequences, and the like; during modification of the viral genome, the method helps to improve mutagenesis efficiency, accurately control DNA virus for genome site-directed mutagenesis and specific gene knockout, simplify operation steps of inserting the DNA virus carrier by an exogenous gene, and improve efficiency that the exogenous gene is integrated to the viral genome, so that the work of screening high-flux recombination viruses is convenient to conduct.

Description

technical field [0001] The invention relates to a DNA virus genome modification method, in particular to a DNA virus genome modification method at a fixed point. Background technique [0002] At present, there are mainly two methods for the site-directed mutation of the DNA virus genome. One method is to carry out extracellular gene recombination operations on the virus genome with the help of tool enzymes such as restriction endonucleases, but for larger virus genomes, it is difficult Find restriction enzyme sites that can be used. Another method is to use the intracellular homologous recombination system to transfect the homologous sequence similar to the viral genome into the virus-infected cells to complete the targeted editing of the genome sequence in a specific region, because the homologous recombination efficiency is extremely high Low, so it is not suitable for high-throughput screening of virus vaccine strains. In order to overcome this difficulty, it is often ne...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/33
Inventor 寸韡李琦涵毕研伟孙乐高丹丹丁晨李智华肖红剑闫玲梅
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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