Construction Of Chimeric Calicivirus And Prokaryotic Expression Of FCV Protective Epitopes

It is much similar in form,structure, genome size, structure and function of genes between RHDV and FCV, which belonged to calicivirus. But it is significant differences in their biological characteristics, especially in the ability of adaptation to cell in vitro. The FCV can be cultivated in a variety of primary or subcultured cells, while RHDV can not. So, the expression of RHDV capsid protein was studied by using FCV as carrier. First, FCV genome infection clone and chimeric virus plasmid was constructed. The objective fragments were amplified from pFCVm-4505 and pUC18 including objective fragments, digested and linked to form pUC-FCV′. Then the VP60 gene of RHDV was inserted into pUC-FCV′to form pUC-FCV′-VP60.At the same time, the plasmid was constructured including whole length FCV with a mutation site 5121 and pUC18, digested with MluI and NcoI, and linked with VP60 which also had been digested with MluI and NcoI to form pUC-FCVm-VP60.After digested with SmaI and NcoI, it was transcripted using vitro transcription kit. Then the product was carried into F81 cell through Lipofectamine.The expression of RHDV VP60 was determined by indirect immunofluorescence after 20 hours of transfection. After repeated experiment, it can conclude that show that the VP60 can be expressed in reconbinant vrius, but can not stable exsiting. Construction and expression of pET-FCV The recombinant plasmids pET-FCV was constructed by cloning gene into pET-28a(+) and expressed in BL21(DE3).The recombinant proteins were highly expressed in inclusion bodies which was induced by IPTG. The yield of recombinant protein was up to 20% of the total bacterial protein and was determined by Western blotting. The indirect ELISA was established to detect serum anti FCV antibodies using the recombinant fusion protein as antigen and goat anti-cat IgG HRP conjugate as the second antibody. The best conditions were that coating antigen , 1μg per well, blocking with 1% fetal calf serum and washing with the supernatant of normal E.coli lysate.It is a simplicity,rapidity and economical cost assay method.