Construction Of Recombinant Pseudorabies Virus-Porcine Circovirus Type 2 Live Vaccine SA215(C) Strain And Study On Some Of Its Biological Characteristics

Porcine circovirus type 2 is one of the new animal viruses in recent years. It is associated with various disease syndromes in pigs, especially post-weaning multisystemic wasting syndrome. This disease first emerged in West Canada in 1991. Clinical symptoms include progressive weight loss, respiratory disorders, fever, pallor and jaundice. Pathological and serological survey show that PMWS is widespread in many countries by and has caused serious economic losses of the swine industry worldwide. To develop safe and effective vaccine to prevent and control PCV2 infection in swine, PCV2 ORF2 gene vaccines and PCV2-PRV bivalent genetic engineering vaccines against porcine circovirus type 2 and pseudorabies virus were studied and the following researches were done.1. Cloning of PCV2 ORF2 gene and its expressing in ST cellsWith PCR method, the complete coding region of PCV2 ORF2 gene was extended from plasmid pMD18-T-ORF2 containing the genetic sequence PCV2 ORF2. PCR product of ORF2 gene was cloned into pMD18-T simple vector carrier to generate the recombinant plasmid pMD18-702*. There was not any base substitution mutation of the gene. The plasmid pMD18-702* was double digested with HindIII and Kpn I and then the fragment PCV2 ORF2 gene was extracted and then inserted into cell expression carrier pcDNA3. 1 (+) of mammal to construct eukaryotic recombinant plasmid pcDNA-ORF2 expressing PCV2 ORF2 gene. After the plasmid was transiently transfected to ST cells in vitro, it was proved that recombinant plasmid pcDNA-ORF2 could be transcripted in ST cells of mammal and the fusion protein was identical to the predicted size and could have reaction with PCV2 standard positive serum, which indicated that the fusion protein possessed immunological activity.2. Construction of recombinant pseudorabies viruses expressing PCV2 ORF2 geneORF2 whole gene extended with PCR method was inserted into multi-cloning site of PRV general transfer carrier pPI-2. EGFP to construct transfer plasmid pPI-2.EGFP.ORF2. And then this plasmid was transfected into ST cells with genetic sequence PRV SA215. When cytopathic effect occurred in cells, recombinant virus SA215(C) was obtained from the cytopathic cells by plaque purification. The recombinant virus was identified with Southern blotting or PCR and expression of extraneous gene in recombinant virus was detected with Western blotting or RT-PCR. The results showed that the construction...