Construction Of PRV UL21-defeated Strain With Bacterial Artificial Chromosome And Studies On Biological Characteristics

Pseudorabies virus,also called Aujeszky’s disease virus or Suid herpesvirus 1,belonging to Herpesviridae,Alphaherpesvirinae,is the pathogen of Aujeszky’s disease distributed throughout the world.The hosts of PRV include almost all kinds of mammals except higher primates,and its main natural hosts are pigs.PRV triggers the respiratory inflammation,abortion,stillbirth of pregnant pig and the death of piglet,which causes severe economic losses every year in China.The genome of PRV contains about 150 kb,consisting of the unique long region,the unique short region,the inner repeated sequence and the terminal repeated sequence.The genome of PRV encodes at least 70 proteins,including structural proteins and nonstructural proteins.pUL21 is a conservative inner tegument protein probably involved in maintaining the virulence of herpesvirus.The UL21 gene is thought to be involved in the virion’s packaging.The UL21 defected PRV shows a higher ratio of genome concatemers and empty nucleocapsid than wild type.Recent study showed that repair of the UL21 locus in Pseudorabies Virus Bartha enhances the kinetics of retrograde,transneuronal infection in vitro and in vivo,indicating pUL21 is possibly involved in PRV’s axonal transport.Bacteria artificial chromosome is a vector based on natural plasmid F factor in E.coli which has the ability to contain large DNA fragment.Once being established,BAC was applied to constructing the eukaryotic library.BAC library is characterized by high stability,large capacity,high speed of replication,and easy to mutant.In 1997,BAC was firstly applied to cloning the genome of mouse cytomegalovirus [1].The study of herpesvirus comes to a new age.Because methods of prokaryotic genome engineering can be used to edit virus’ genome by BAC,which saves much time and labor,more and more infectious clones of herpesvirus were constructed to edit their genome efficiently.In this study we constructed the UL21-defected PRV and reverse mutant to know more about the affection that UL21 deficiency has on the replication of PRV and the efficiency of editing PRV genome by BAC.To knock out UL21,the Kan cassette was amplified and then electroporated into GS1783 containing pPRV Ea,to accomplish the first Red combination.Then I-SceⅠ and Red operon was induced to finish the second Red recombination.The PCR identification showed that the efficiency of Red recombination was not stable.But the positive clones can be got anyway,which was not disturbed by the plasmid from PCR template,indicating PRV mutant can be constructed with BAC in a short time,which will save much time and labor.Finally,the result of western blot indicated that rPRV-ΔUL21 lost the ability to express pUL21,and we succeeded to get UL21 defected PRV.The plaque size comparison experiment showed that the plaque size of rPRV-ΔUL21 on MDBK cells was slightly but remarkably smaller than PRV Ea.This result indicated that UL21 is substantially involved in the replication of PRV Ea.However,the function of UL21 is not necessary to PRV replication.To make sure that the mutation of UL21 didn’t have interference with the overlapped lncRNA CTO-L,real time PCR was conducted to quantify the expression of CTO-L after PK-15 cells were infected by PRV.The result showed that the point mutation of UL21 hardly affects both of CTO-L’s sequence and expression level.In this study we constructed PRV mutant with PRV BAC,and found the optimal condition,to make it convenient for others to construct PRV mutant in the future.The study on biology character of rPRV-ΔUL21 gives an insight into the function of UL21 gene,and reveals the relationship between UL21 and CTO-L.