Establishment of an Efficient and Flexible Genetic Manipulation Platform Based on a Fosmid Library for Rapid Generation of Recombinant Pseudorabies Virus.

ABSTRACT: Conventional genetic engineering of pseudorabies virus (PRV) is essentially based on homologous recombination or bacterial artificial chromosome. However, these techniques require multiple plaque purification, which is labor-intensive and time-consuming. The aim of the present study was to develop an efficient, direct, and flexible genetic manipulation platform for PRV. To this end, the PRV genomic DNA was extracted from purified PRV virions and sheared into approximately 30-45-kb DNA fragments. After end-blunting and phosphorylation, the DNA fragments were separated by pulsed-field gel electrophoresis, the recovered DNA fragments were inserted into the cloning-ready fosmids. The fosmids were then transformed into Escherichia coli and selected clones were end-sequenced for full-leng

SUBMITTER: Zhou M 

PROVIDER: S-EPMC6133995 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature