Construction And In Vitro Reproduction Feature Of An Infections Clone Of Pseudorabies Virus Strain Fa Genome Maintained As A Bacterial Artificial Chromosome

Pseudorabies virus(PRV),or “Aujeszky”s virus,is a member of alpha herpesvirinae,Herpesviridae,which can cause pseudorabies disease in swine,bovine,sheep and wild animals.Pseudorabies is an acute infection diease and characterized by high fever,extreme itching(except swine)and encephalomyelitis.It was classified as the second animal infectious disease by the World Organization for Animal Health(OIE)and extremely difficult to prevention and have caused great economic losses in animal husbandry especially the intensive pig industry.PRV gene is a double-stranded DNA of approximately 150 kb and encodes 70-100 proteins,mature virus particles contains about 50 kinds of proteins.For a long time,the main method studying on the functional genes of herpesvirus was to construct genetic-deleting mutants.However,it was tedious to produce recombinant mutants and have the feature of genetic instability using traditional homologous recombination method which limited the study of herpesvirus.But in 1999,Gregoory et al,American researchers,successfully constructed a Full-Length infectious Clone of PRV based on bacterial artificial chromosomes(BACs).Sinece then,BAC technology greatly facilitate the study of herpesvirus and provide a new way to develop the high efficiency vector system.In this study,a transfer vector p UC-Fa TKAM-GFP-gpt-Fa TKB was constructed by inserting the EGFP expression cassette and gpt gene with Loxp site and BAC plasmid p Belo BAC11 between two homologous arms containing partial TK gene of PRV Fa strain and a recombinant virus(r PRV-Fa-BAC)was achieved by cotransfecting the transfer vector and PRV genome into Vero cell.After 3 passages of gpt selection and 4 passages of phage assay based on GFP expression,the recombinant virus(r PRV-Fa-BAC)contain BAC plasmid was purified.The circular genome DNA of the recombinant virus was extracted timely after infected Vero cell,then transformed into E.coli DH10 B competent cell and a PRV BAC clone was identified by Bam H Ⅰ digestion.The recombinant virus(v PRV-BAC-Fa)were reconstituted after the positive PRV BAC clone was transfected into Vero cells.After four passages on Vero cell,the GFP was expressed stably,Genes in the BAC plasmid or genes on the virus genome adjacent to the insertion locus could be amplified by PCR,which indicated that v PRV-BAC-Fa can be passed down stably.The reproduction feature of v PRV-BAC-Fa in vitro was investigated by phage assay and single-step growth curve mensuration,the results showed that the reproduction rate of vPRV-BAC-Fa were slower about 4 hours than parental virus PRV Fa P8,which indicated that the delection of TK gene make the reproduction rate of v PRV-BAC-Fa in vitro culture slower.The successful construction of bacterial chromosome infectious clone of PRV Fa strain was not only lay a solid foundation for developing a candidate PRV gene-deleted vaccine,but also have a profound effect on the establishment of reverse genetic manipulation platform of PRV.