Pseudorabies virus genome fosmid library, construction method and application in construction of mutants

Abstract

The invention discloses a pseudorabies virus (PRV) genome Fosmid library, a construction method and its application in the construction of PRV mutants. The present invention constructs the PRVTJ strain genome Fosmid library, selects 10 groups of 5-cosmid systems that can cover the complete PRV genome and have overlapping regions, screens out 5 groups of cosmids that can rescue the complete PRV, and identifies the best cosmid system. The cosmid combination was used to construct a recombinant PRV fused with VP26 to express EGFP. There was no difference between the reporter virus and the parental virus in titer and growth characteristics. The invention divides the huge PRV genome into several small fragments to construct a Fosmid library, which improves the stability of the library, makes the transformation and modification of the PRV gene simpler and more convenient, and shortens the cycle of building the library. The invention lays a foundation for rapid and accurate modification of the PRV genome, construction of recombinant vaccines and the like.

Description

technical field [0001] The present invention relates to a Fosmid library and a construction method thereof, in particular to a pseudorabies virus genome Fosmid library and a construction method thereof. The present invention further relates to the application of the pseudorabies virus genome Fosmid library in rapidly constructing pseudorabies virus mutants, which belongs to pseudorabies virus Genomic Fosmid library and its construction and application fields. Background technique [0002] Pseudorabies (Pseudorabies, PR) is a kind of pig, sheep, cattle and other livestock and wild animals caused by pseudorabies virus (Pseudorabies virus, PRV), with fever, itching (except pigs), encephalomyelitis, respiratory Acute infectious disease characterized by neurological and nervous system disorders (Mettenleiter, 1989). Pigs are the storage host and main source of infection of the disease, which can be transmitted horizontally through the digestive tract and respiratory tract, and c...

AI Technical Summary

Problems solved by technology

However, the PRV genome is as high as 143kb, and it is very easy to break. Once the genome is broken, it loses infectivity and cannot obtain recombinant virus

In addition, the entire genome of PRV is connected with the BAC plasmid, and fragment deletions often occur during the electrotransformation process, which affects the efficiency of obtaining recombinant viruses (Lerma L, AL,Wagner S,Dinu M,Martín B,Tabarés E.Construction of recombinant pseudorabies viruses by using PRV BACsdeficient in IE180or pac sequences:Application of vBAC90D recombinant virus to production of PRV amplicons.Virus Res.2016,213:274,-282.) Therefore, the efficiency of obtaining PRV mutants by traditional homologous recombination methods is very low and takes a long time

Images