Method for improving homologous recombination efficiency and recombination virus screening of pseudorabies virus

Abstract

The invention discloses a method for improving homologous recombination efficiency and recombination virus screening of pseudorabies virus. According to the method, a pseudorabies virus AH strain transfer vector containing an EGFP gene complete expression box is subjected to enzyme digestion linearization, and then subjected to homologous recombination with the pseudorabies virus AH strain; and the obtained recombination virus screening method comprises the steps of separating fluorescent single host cells with pathological changes, and then performing further screening by improved plaque experiment. After recombinant plasmid enzyme is subjected to digestion linearization, the enzyme is subjected to cell transfection; next, virus homologous recombination is performed, so that the homologous recombination efficiency is improved; by virtue of the method in combination with single cell absorption and plaque purification, the screening and purifying time of the recombination viruses is greatly shortened; and therefore, the method is of great significance to improvement of the virus homologous recombination efficiency, has high application prospect in single cell separation, particularly recombination virus screening, and can be widely applied to the recombination virus screening.

Description

technical field [0001] The invention relates to a method for improving homologous recombination efficiency of pseudorabies virus and screening recombinant viruses. More specifically, it involves how to increase the chance of homologous recombination between virus and plasmid in cells, and how to purify recombinant virus by using an inverted fluorescence microscope to separate single cells and plaque screening. Background technique [0002] Pseudorabies virus (Pseudorabies viurs, PRV) belongs to Herpesviridae (Herpesviridae), α-herpesviridae subfamily (Alpha-herpesviridae), and can cause most animal infections. Porcine pseudorabies is a viral disease caused by pseudorabies virus, which was first discovered in the United States. At present, countries such as Europe and the United States have basically eliminated pseudorabies. However, in 2011, the domestic PRV epidemic suddenly broke out on a large scale. The traditional gE-deficient vaccine has been proven to be unable to pr...

AI Technical Summary

Problems solved by technology

However, flow cytometry has high requirements for instruments, complicated operation and high cost, and cannot perform grade separation according to the fluorescence intensity of cells.

Simply using plaque screening often takes a long time and delays the progress of research

Traditional plaque screening requires the use of phenol-free erythrocyte medium, and neutral red staining is required to identify viral plaques, and neutral red is toxic when it is decomposed by light, and it will cause damage to the virus in practice

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