Pseudorabies virus genomic Fosmid library, construction method and application thereof in constructing mutants

Abstract

The present invention discloses a pseudorabies virus (PRV) genomic Fosmid library, a construction method thereof and application thereof in constructing PRV mutants. A genomic Fosmid library of a PRVTJ strain is constructed, 10 sets of 5 cosmid systems capable of covering a complete PRV genome and having mutual overlapping regions are selected, 5 sets of cosmids which can rescue intact PRV are screened, a best cosmid combination can be identified, the best cosmid combination is used to construct a recombinant PRV which expresses EGFP by in fusion with VP26, and the reporter virus and a parental virus do not differ in titer and growth characteristics. The large PRV genome is divided into a plurality of small fragments to construct the Fosmid library, the stability of the library is improved, PRV gene transformation and modification are simpler and more convenient, and the constructing cycle of the library is shortened. A foundation is laid for rapid and accurate modification of the PRVgenome and construction of a recombinant vaccine.

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...

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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

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