Study On The Exogenous Gene Inserted Site In The FCV Genome

The F81 cells were infected with FCV to generate the virus.The virus-oriented RNA was extracted from the FCV with Trizol.Three pairs of primers specific to the whole genome neculeotide sequences of the FCV-CH-GD were designed and synthesised.The three fragments produced by PCR were cloned into T vector,namely PL,PR and pUC2634,respectively.Then the three fragments were subcloned into a modificated pUC18 vector,namely pUCFCV.The vector was lineared by restricted enzyme SphI.The linear vector transcripted in vitro .There is not significant path-ological difference between F81cells infected with transcription or wild FCV.It was manifestated that the infectious clone had been construced successfully via RT-PCR and restricted enzyme MluI .Then a transfer vector was construced.There were two kinds of this vector.One kind is that three fragments were amplificated from different site of thePUC2634 .Three Transfer vectors were construced with GFP gene and one of the three frag ments.Exogenious GFP gene was thrusted into one of three sites in the pUC2634 ,and then three vector inserted with GFP were constructed.An- other one kind is that two fragments were amplificated from the pUC2634,GFP gene took place one region of the fragment respectively. And two transfer vectors were constructed.These vectors and pUCFCV were double digested by MluI and SphI .The GFP gene was inserted in different sites of the FCV genome.The five constructed plasmids were digested by NcoI and transcripted in vitro.The transcriptions were used to infected the F81 cells .The inserted GFP gene cough be expressed but not the substituted GFP gene.The cells infected with transcription which GFP gene was linked to the non-constructal protein gene or lead protein gene didn't have any pathological changes after four genetations .In the case of caspid protein gene ,pathological changes appeared. After cells'four generations ,the GFP gene cough not be detected with the RT-PCR in the case that it was linked to the non-constructral protein gene or the caspid protein gene,but the result was positive in the case of lead protein gene.The GFP gene was detected with RT-PCR only in the case that it was linked to the caspid protein gene or lead protein gene .Accordingly,it was confirmed that exogenous gene cough be inserted to the caspid protein gene or lead protein gene ,but the exogenous gene cough not replace any gene in the genome.The study suggested that it was possible that non-GFP exogenous gene was inserted into the FCV vector.