Genome Sequencing And Construction Of Infectious Clone Of The Foot-and-mouth Disease Virus O/Akesu/58 CE39A Strain

At present,the whole-virus vaccines were applied to prevent and control FMD in our country.However,the manufacturing process of this vaccine is complicated also has risk of escape of live virus,which can cause the epidemic by virus inactivation in the vaccine does not completely,but also has shortcomings such as short duration of immunization and common side effects,result in hardly satisfy the requirement for the healthy and stable development of animal husbandry.Genetic engineering is expected to overcome the above shortcomings and produce vaccines with safer,more effective and longer immune duration,especially gene-deleted virus vaccines.To achieve this purpose,used the FMDV strain O/Akesu/58 CE39 as the research target strain,the strain was weakened by The chicken embryo(chick embryo,CE)that was once used for the foot-and-mouth disease attenuated vaccine,which was sub-cultured by BHK-21 cells,and indirect immunofluorescence test was used to detect its pathological features,and a clone O/Akesu/58 CE39 A was chosen out by the plaque selection experiment.Based on a reference genome(Gen Bank accessory number AJ539138)of FMDV,O serotype,primer pairs were designed and synthesized to amplify seven overlapped fragments of the clone.The 5'-UTR region was amplified by 5'-RACE method,and other fragments were amplified by generally PCR.All fragments were cloned into p MD19-T vector for sequencing.After redundant sequence eliminated,seven fragment sequences were spliced sequentially to get real sequences of genome of the O/Akesu/58 CE39 A strain.Experiment results showed that the full length of the virus genome is 8223 nt,among which5'NCR is about 1101 nt-length,leader protein L coding region is about 603 nt-length,3'NCR is about 98nt-length and 3' End of poly(A)tailing structure is about 25 nt.Full length Coding region is about 6999 nt,which can be translated to a polyprotein including 2333 amino acids.Sequence analysis showed that the strain belonged to the Cathy topological type.Furthermore,in this study,the optimal conditions for successfully amplifying the FMDV genome's5'-end sequences by general PCR method were explored.In this experiment,72 PCR was carried out,and the success rate was 6/72,which indicated that the amplification of FMDV 5' terminal sequences was difficult,and the inversion of the primers and amplification primers should be rationally designed.The real full-length genome of O/Akesu/58 CE39 A was divided into six fragments to amplify,digest and clone into p UC57 vector successively to construct full-length infectious clone.The constructed FMDV genome full-length plasmid p UC57-O/Akesu/58 CE39 A,digested with Eco RV,appear theexpected size band 11041 bp.Then,the plasmid,digested with Sal I,break up into two expected size bands 2761 bp and 8269 bp.The results of enzyme digestion showed that the full-length plasmid of FMDV genome was successfully constructed,which provides the foundation for the rescue of the virus and the reverse genetics of many RNA viruses.