Generation Of A GE/TK-Gene-Deleted Mutant Derived From Pseudorabie Virus Bartha-K61 Strain

Pseudorabies virus (PrV) is the causative agent of Aujeszky's disease, which results in significant losses in pig husbandry. Thymidine kinase (TK) gene is one of the main virulent genes of PrV, and it is essential to the propagation of PrV in the nerve tissues, but dispensable for virus replication and infection in other tissues.In this study, a 1.7kb KpnI fragment and a LacZ gene expression cassette carrying the E. coli LacZ gene under the control of SV40 promoter were inserted into the transfer vector pBdTK-Uni (a 277bp AccI-Accl fragment in the TK gene was deleted). The new transfer vector was called pUni-LacZ. The transfer vector pUni-LacZ and purified genomic DNA of strain Bartha-K61 were used to cotransfect Vero cells using lipofectin transfection procedure. After obvious cytopathogenic effects developed, virus-contained supematants were harvested, and the progeny viruses were screened for LacZ-expressing viruses by a plaque assay using X-gal. Single blue plaques were picked, and a recombinant PrV stably expressing LacZ gene (designated as rPrV-LacZ) was obtained after ten cycles of plague purification and PCR identification.The results showed that the LacZ gene expression cassette was stably expressed in the recombinant rPrV-LacZ derived from Bartha-K61 strain. Compared to its parental virus Bartha-K61 strain, it displayed no obvious differences in virus multiplication and cytopathogenic effects in different cell cultures (PK-15, IBRS2, Vero and CEF). The transfer vector pBdTK-Uni can be used for the construction of recombinant PrV expressing foreign gene(s).Postgraduate: TianZhijunSpecialty: Preventive Veterinary Science Supervisors: Prof. Li YijingProf. Tong Guangzhi...