Sequence Analysis And Infectious Clone Construction Of Feline Calicivirus

In this study three pairs of specifical primers of FCV gene were designed according to the sequences from GenBank by Premier 5.0.Complete sequence of FCV CH-GD strain were amplified by RT-PCR (Reverse Transcription Polymerase Chain Reaction) and recombinant PCR.DNAStar software was used to analyse nucleotide sequences and amino acid sequences. The homology of CH-GD strain which was isolated first in Guang Dong Province of China with reference strains was between 75.4%~77.1%. It was lower than that was between reference strains(78.9%~99.9%). There were two branches between 14 strains.CH-GD strain was in one branch and there was no evident difference in district .The homology of amino acid and molecular evolution of three ORFs showed that it was higher than complete nucleotide in homology of nucleotide of isolated strains.EA and ES which were separated in the position of 961 and 1072 were completely identical with isolated strains. ED was located in 686 which was identical with most reference strains. There were clearly difference in position of 47,332 and 1364 compared with othe reference strains which was showed that the variation had occrued in CH-GD strain.The effection of variation to the characteristic and function need to be studied further.Analysing six region of FCV capsid protein, the characteristic of A~F region was equal to that had been reported. Antigenicity and hydrophilicity were different from reference strains in three region where three consecutive amino acid were variation. In order to determine convenience,the new primers within a artificial mutant site,two enzymic hydrolysis position and T7 promoter were designed according to the sequences of CH-GD strain.The new PCR product of CH-GD strain was obtained by new primers.The plasmid P′FCV+T including new PCR product was transcripted and then transfected to F81 cell, at last tested by RT- PCR and enzymic hydrolysis.The result showed that infective FCV RNA was expressed in F81 cell and mutant site was detected.The construction of infectious clone of FCV was successful.This study elucidated the molecular evolution of FCV and will provide a basis for the study of biology characteristic and pathogensis of FCV.The successful construction of FCV infectious clone has laid a foundation for the study on Calicivirus chimeric vector vaccine.