Construction of Recombinant Marek's Disease Virus Expressing the NDV-F gene and its Replication in Chickens and in Vitro / 病毒学报

ABSTRACT

We used a meq-deleted attenuated MDV-I strain GX0101Δmeq as a vector to construct a recombinant virus expressing the exogenous gene NDV-F. The ORF of exogenous gene NDV-F was inserted into the eukaryotic expression vector pcDNA3.1(-). Then, the expression cassette of NDV-F which contains the CMV promoter was amplified. Simultaneously, we amplified the selected gene Kan+ expression cassette and inserted them into the PMD18-T vector. Tandem expression cassettes were amplified using primers containing the 50-bp homologous arm of MDV-US2. The PCR product was electroporated into EL250 host bacteria containing GX0101Δmeq. Then, the Kan+ expression cassette was deleted from the recombinant virus genome using 1% arabinose. The plasmid of the positive clone which the Kan+ expression cassette was deleted was extracted and transfected into CEFs to rescue the recombinant virus. The recombinant virus was injected into chickens to observe its growth and replication. The recombinant virus rMDV-F containing the exogenous gene NDV-F was rescued successfully. The recombinant virus could duplicate and express well in CEFs, and grow and replicate well in chickens. Using GX0101Δmeq as a vector, combined with a recombinant system of Red E/T and FLP/FRT, we constructed a recombinant virus that expressed the exogenous gene NDV-F. This study could lay the foundation for further study of recombinant viruses.