Establishment Of Reverse Genetics System For Feline Calicivirus And Expressing Of GFP

Feline calicivirus ( FCV) infection contained of diseases such as upper resp iratory tract, acute mouth ulceration and chronic stomostitis and acute arthritis in cats was caused by FCV.Feline Calicivirus were first isolated by Fastier in 1957, then it was reported that Feline Calicivirus were isolated from feline and cheetah in most countries.Nearly all felid were susceptible to Feline Calicivirus.The family of Calicivirus includes important pathogens of men and animals.In addition to Feline Calicivirus could endanger full felid,rabbit hemorrhagic disease virus could lead to tremendous economic loss of the rabbit industry.Human Calicivirus,for example,the Norwalk virus can cause infectious gastroenteritis in people,especially infants and young children.However,the majority of Calicivirus could not be cultured in cells,that limited the development of research in Calicivirus.Except vesicular exanthema of swine virus,FCV is unique to culture in cells in the famliy of Calicivirus.So ti is a good ider to understand the bionomics,genome structure and function and recombinant virus vaccine of Calicivirus by research the FCV.The F81 cells were infected with FCV to generate the virus,then the virus-oriented RNA was extracted from the cells with Trizol.The whole genome neculeotide sequences of the FCV-CH-GD strain were amplified by RT-PCR,the two fragments produced by PCR were cloned into T vector, which named pFCV5′-3200 and pFCV3′-4800 respectively.It need three steps to introduce Hammerhead ribozyme(HamRZ) at the 5′end of the full-length genome cDNA by PCR.Then the two fragments were cloned downstream of the CMV promoter of the veetor pcDNA-HdvRz.After Purified,the recombinant infectious clones named pFCV-HdvRz was directly transfected F81 cell indued by LipofectamineTM2000(Invitrogen).48h after transfection,the supernatant of cell cuture was harvested and continue passaged in F81 cell.The results of indirect immunofluorescence and RT-PCR all verified that FCV was reseued successfully.The RNA PolymeraseⅡ-based reverse genetics system has laid a foundaion for the gene function research and construction of recombinant virus-based FCV vaccine.Transfer vector was construced with GFP gene and the whole fragment amplificated from vector pFCV3′-4800.Both this vector and pFCV-HdvRz were double digested by HindIII and NotI and then linked.the recombinant infectious clones was construced,which GFP gene was linked to caspid protein gene of the 3′end containing 3C-liked proteolytic enzyme geme at two endpoint,namely pFCV-GFP.When this vector directly transfected F81 cell, the cells appeared pathological changes after six genetations,the green fluorescent could be directly observed by fluorescence microscope and GFP could be detected by RT-PCR.The green fluorescent protein (GFP) was expressed by utilizing the character of FCV adapted to F81 cell and the RNA PolymeraseⅡ-based reverse genetics system of FCV,and it was laid a foundation to express the gene of anthor Calicivirus,such as RHDV,HuCV,HEV and so on.