Study On The Construction And Expression Characteirstics Of Canine Distemper DNA Vaccine Based On SFV-replicon

Canine distenper is a highyf contagious viral disease caused by the canine distenpervirus(CDV)，its prevention is mainyf dependent on the live attenuate vaccines. However, liveattenuate vaccines have the disadvantages of reverting to vinience among siuroiuiding animals，susceptible to maternal antibody and can not differentiate inefcted rfom vaccinated animals.Therefore, it is necessary to develop a new generation of the nucleic acid vaccine to replace the liveattenuate vaccines.To inprove the immiuie effect of canine distenper DNA vaccine, in this research we used thea^)havirus replicon vector pSCAl as a ifuidmental vector which had the characteristics of selfreplication and eiffcient^egress ofreign proteins,and the bovine herpes virus type I VP22proteinwith transduction tfinction and CDV isolaets of F gene as the target genes^we constructed theeukaryotic expression plasmid that harboired the target genes pSCAl-f and pSCAl-VP22-F.Finally, the recombinant plasmids named pSCAl-F and pSCAl-VP22-F were constructedand identiifed by restriciton enzyme anayfsis and sequencing. The recombinant plasmids weretransfected into BHK-21cells by using X-tremeGENE9DNA Transefction Reagent, IF A andWestern blotting were performed to detect F expressions, these assay resists showed that (1) theRT-PCR products of electrophoresis corresponded to the expected size；(2) BHK-21cellstransfected with the two recombinant plasmids appeared lfuorescence，the recombinant plasmidpSCAl VP22-F，s lfuorescence was more obvious than the recombinant plasmid pSCAl-F’s,while the enpty plasmid without lfuorescence;(3) the westenr blotting immiuioreactive bands of thetwo recombinant plasmids corresponded to the expected size62kDa, and the recombinant plasmidpSCAl VP22-F band was more obvious;(4) The BHK-21cells transefcted with the tworecombinant plasmids were stained with Hoechst33258,then we could see the chromatin pyknosisphenomenon under the lfuorescence microscope which was similar to the positive control (apoptosisinducers).In this study we conifrmed that recombinant plasmids pSCAl-F and pSCAl-VP22-F hadgood antigenicity and they coiid express in the transefcted BHK-21cells;Fiu*thermore, Apopotsisassay conifrmed that recombinant plasmids can induce the transfected cells apoptosis.In terms of theIF A and Western blotting resists, pSCAl-VP22-F was better than pSCAl-F，suggested that VP22can enhance its ifision protein CDV F protein transport between cells.