Cloning, Sequencing And Constructing Eukaryotic Expression Plasmid Of The GE Gene Of Pseudorabies Virus

The reference Min-A strain of pseudorabies virus(PRV) was reproduced by adaption to PK-15 cell and genome DNA was extracted from the harvested infected culture after ultrasonification and centrifugation as template. A pair of primers were designed and synthesized based on the published gE gene sequence of PRV-Rice strain for amplifying gE gene of PRV Min-A, yielding a 1.7kb band. The segment was linked to pUC19 plasma DNA by means of T4 DNA ligase, transformed into E.coli JM109 permissive cells, and incubated on LB fray containg Amp, X-gal and IPTG. Small amount of plasma was extracted by base cleavaging for enzyme digest analysis and PCR, resulting in recombinant plasma pUgE DNA containing PRV gE.The recombinant plasmid pUgE DNA and transfer vector pFastBacl DNA were treated again in the same enzyme,were linked by means of T4 DNA ligase and transformed into E.coli JM109 permissive cells, yielding recombinant transfer vector plasmid pFastBac-gE DNA and were transformed into DHlOBac containing vector Bacmid. The recombinant Bacmid DNA was extracted from the yielding white bacterid colonies by tranferated. For enzyme restriction identification, designated as rBacmid-gE DNA. Insect cell sf9 was transinfected with the purified rBacmid-gE DNA by Ca3(PO4)2 precipitation. The transinfected supernant was collected and centrifugated at low speed, and the supernant was recombinant virus named rvBac-gE. SDS-PAGE analysis of PCR product of the recombinant virus showed with the amplified fregment was in conformity with the expected size, suggesting correct recombination and cloning of the gE gene.