Establishment Of Feline Calicivirus (FCV) Reverse Genetics System Using RNA Polymerase Ⅱ And Expression Of RHDV VP60 Gene

Using reverse genetics systems for RNA virus rescue and manipulating in DNA level is a powerful technique in researching viral molecular biology and interactions between RNA virus and host. All human caliciviruses and the members of the genus Lagovirus can not be propagated in standard tissue culture systems. In contrast, vesiviruses replicate to high titers in cell lines. The best-studied member of the genus Vesivirus is FCV. This study selected Feline Calicivirus which was the pathogen of vesivirus as a model on the basis of obtained full-length cDNA clone of FCV CH-GD strain and attempted to construct FCV reverse genetics systems and rescue molecular marked FCV.1. Construction of full-length genome cDNA clone of FCV CH-GD strainPrimers were designed for sequencing the genome of CH-GD strain. The viral full-length genome cDNA sequence was obtained from plasmid pUC-FCV by PCR. A molecular marker(Mlu I enzyme site) was introduced to distinguish wild-type virus and rescue virus. Remake the pCDNA vector by PCR and inducing ribozyme into the genome 3'of pCDNA. Every amplified fragments and reconstructive pCDNA vector were digested by restriction enzyme to construct full-length genome cDNA (pCDNA-HDVRZ-FCV) clone of CH-GD strain. The pCDNA-HDVRZ-FCV was indicate by PCR, digested by restricton enzyme and sequenced.The results indicated that the construction of full-length cDNA clone of FCV CH-GD strain is successful. The whole genome sequence, ribozyme sequence and a molecular marker(Mlu I enzyme site) are checked in pCDNA-HDVRZ-FCV clone.2. Identification of rescued FCVpCDNA-HDVRZ-FCV used as a template synthesized in vivo transcription by means of RNA polymeraseⅡsystem. F81 cell was transfected with plasmid DNA using lipofecta-mine reagent, typical FCV CPE would be seen after 3 generations. RT-PCR was used for detection. All the detection results showed that rescued virus was obtained and construction of infectious cDNA clone of FCV was successful. The infection of wild-type virus was excluded through detection of molecular marker of infectious clone.3. Expression of RHDV VP60 in F81 cellThe pCDNA-HDVRZ-FCV and two transfer vectors (pFCV-O and pFCV-V) are double digested by Kpn I and Not I. The RHDV VP60 gene was inserted into two different sites of FCV genome (after LC and after VP1). Then, the chimeric FCV was rescued after transfecting plasmid into F81 cell through Lipofectamine.The expression of RHDV VP60 was determined by indirect immunofluorescence after transfected 48 hours. Continuous cell culture indicates that the immunofluorescence thin down and can not be detected after 7 generations, this shows that the VP60 can be expressed in chimeric vrius, but is not infectious.In a word, FCV reverse genetics can be used to research functional genomics, molecular mechanism of pathogenesis and variation of FCV and show some prospects in researching of gene knocked-out vaccine and marker vaccine.