Construction Of PTV Swine/CH/IMH/03 Full-length CDNA Clone And Expression Of 3D Protein

Porcine teschovirus (PTV) was described as causative agents of severe and mild neurologicaldisorders known as Teschen/Talfan disease, reproductive failure, pneumonia, diarrhea, and dermallesions of swine. PTV was classified as member of Teschovirus genus of Picornaviridae on virustaxonomy at theâ…ªth International Congress of Virology (ICTV), Sydney in Australia in 1999.Clinical syndromes caused by different serotypes of PTV were different. Talfan/Teschen diseasewas reported in Teschen and other European countries firstly in 1930s, and its agents was laterdetermined as PTV. Nowadays Talfan/Teschen disease was worldwide, and had brought greateconomic loss. Therefore, study of PTV vaccine, diagnosis and immunologic mechanism had beenconcerned with by more and more researches.Recently, reverse genetics became an effective tool for stuty of viral genetic engineeringvaccine, gene function and pathogenesis mechanism, et ac. In order to construct infectious cloneof PTV, we firstly constructed 4 overlapping subclones covering full-length cDNA. After assembly,pSK-PTVFL containing PTV full-length cDNA was constructed. Determined by sequencing, wefound 5' end of PTV-1 in pSK-PTVFL was poly(C), followed by unique open reading frame, andpoly(A) at the 3' end. Besides Kpnâ… and Sacâ…¡were manually introduced to 5' and 3' of PTV-1expectly, which was used for linearization of plasmid before in-vitro transcription. T7 promotorwas introduced at downstream of Kpnâ… for in-vitro transcription with T7 RNA polymerase.Generally speaking, the full-length clone was: Kpnâ… -T7 promotor-PTV genome-Poly(A)-Sacâ…¡.Compared with parent virus Swine/CH/IMH/03 and othe PTV strains, 5'-UTR and 3'-UTRof this clone had high homology, and were absolutely identical to Talfan, which had gainedintegrity and correct sequences in 5'-UTR and 3'-UTR. ORF of this clone had 3 amino acidsmutations in 2A, 2C and 3D gene respectivly, which could be used as candidate for differentiateengineering virus from parent virus.Meanwhile, 3'-UTR and 5'-UTR of PTV Swine/CH/IMH/03 were cloned, sequenced andaligned, we found 3'-UTR of PTVs possessed less than 100 bases, however, it was extremelyconserved, no more than 10 sites of base substitution. 5'-UTR of PTV Swine/CH/IMH/03 washighly similar to other PTVs too.Based on those upon, we did some work on PTV-1 in-vitro transcriptino, transfection andgained much experience.Successfully construction of PTV-1 full-length cDNA clone and accumulative experienceabout virus rivival lay foundation for study of viral genomic structure, function and anti-virusvaccine based on PTV reverse genetics.In Picomaviridae, 3D protein was RNA dependent RNA polymerase, and had been applied indifferential diagnosis of FMDV infection, and other picornaviruses. In order to establish ansero-assay of PTV for testing antibodies against Talfan/Teschen disease in China, PTV 3D genewas amplified, and then subcloned into pET30a(+). Transformed E.coli BL21 and induced withIPTG, and 3D protein was high level expressed (approximately 66.1%). Western blot results demonstrated that 3D recombinant protein could recognized PTV positive serum specifically andhad a fine immunogenicity. It is possible that 3D could be applied in fast differential diagnosis ofPTV infection and could be helpful for epidemiological survey and prevention of PTV.