A specific site-directed transformation and screening method of DNA virus genome

Abstract

The invention provides a site-specific modification and screening method for a specific DNA (deoxyribonucleic acid) viral genome. The method comprises the following steps:1, establishing site-specific cleavage single-strand and duplex nuclease systems and a homologous sequence; 2, introducing cytocidal infection DNA viruses of the site-specific cleavage single-strand nuclease system and the homologous sequence, and collecting P1 progeny viruses after cells are pathologically changed; 3, introducing cytocidal infection P1 progeny viruses of the site-specific cleavage duplex nuclease system, collecting P2 progeny viruses after the cells are pathologically changed, and separating and purifying the P2 progeny viruses to obtain target progeny viruses, wherein the step 2 is a viral genome homologous recombination step, and the viral genome homologous recombination efficiency is improved by about hundreds of thousands of times under the condition of not introducing random mutation; the step 3 is a specific amplification step, and specific mutation viruses in the progeny viruses are further increased about 10 times. According to the method, mutation types can be effectively controlled, and recombinant viruses with specific mutation, deficiency or intercalation can be rapidly and conveniently screened.

Description

technical field [0001] The invention relates to a DNA virus genome modification method, in particular to a specific DNA virus genome modification and screening method. 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 a single restriction site 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 o...

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Problems solved by technology

[0004] In view of the above-mentioned defects and problems of the prior art, the purpose of the present invention is to provide a specific DNA virus genome fixed-point transformation and screening method to solve the large randomness of the mutation types existing in the existing DNA virus genome fixed-point transformation method , and this random mutation cannot strictly meet the requirements for biosafety, and often cannot meet the needs of precise insertion or deletion of several bases at specific viral genome sites and mutations into specific target sequences during product development.

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