Identification Of The Critical Amino Acids Determining The Dominant Epitope In Non-structural Protein 3B And Construction Of The Negative Marker Foot-and-Mouth-Disease Virus

Foot-and-mouth disease?FMD?is an acute,febrile,and highly contagious disease of livestock caused by foot-and-mouth disease virus?FMDV?.It spreads fast and causes high morbidity.Outbreaks can result in great negative influence on both economy and politics of affected countries.Vaccine inoculation with the conventional inactivated vaccine is the most effective measure to prevent and control FMD at present.However,the conventional inactivated vaccines can not serological distinguish between infected and vaccinated animals?DIVA?,which causes the disease is still widespread in our country.Therefore,it is necessary to develop of improved vaccines with a DIVA property against FMDV.To develop FMD marker vaccine candidate strain with good replication capacity,this paper carried out researchs as the followings:1.We successfully constructed 16 eukaryotic plasmids?S1^S16?expressing the FMDV non-structural protein 3B and 3B mutants by alanine scanning mutagenesis.The recombinant plasmids were transfected into BHK-21cells respectively,then the transfected cells were analyzed by Indirect Immunofluorescence and Western Blot with the monoclonal antibody 3B4B1recognizing the dominant epitope of FMDV 3B protein,which was screened and preserved by our own laboratory.The results showed that the cells transfected only with S12 plasmid can not reacted with the monoclonal antibody 3B4B1,the others can,which indicated that the crucial amino-acid residues recognized by the the monoclonal antibody 3B4B1 are located ²PY-GP⁶ of the3B₁ and 3B₂.2.Using a previous developed FMDV pOZK/93-08 full length infectious clone,we constructed 6 recombinant full length clones containning different amino acid mutant in 3B1 and3B2 proteins.Not I-linearized recombinant plasmids were transfected into BSR/T7 cells expressing T7 RNA polymerase to rescue the recombinant viruses and only three recombinant viruses were successfully recovered.The cell infected with recombinant viruses and wt virus were analyzed by Indirect Immunofluorescence and Western Blot with the monoclonal antibody 3B4B1,the results showed that the virus containing ⁴AGP⁶ to ⁴TAA⁶ in 3B1 and 3B2 can not react with the monoclonal antibody 3B4B1,but the other viruses can,which indicated that the change of ⁴AGP⁶to ⁴TAA⁶ in 3B1 and 3B2 completely abolished the ability of the recombinant FMDV to be recognized by MAb 3B4B1.The recombinant virus conainning ⁴AGP⁶ to ⁴TAA⁶ in 3B1 and 3B2was further analyzed by plaque phenotype,One-step growth curves and genetic stability,the results showed that the recombinant virus has similar growth kinetics and plaque phenotype as the wild type virus and the amino acid mutant nearly does not affect virus replication in BHK-21 cells.Furthermore,the mutant in 3B was genetically stable after 20 serial passages in BHK-21 cells.3.Finally,the prokaryotic expression plasmids of the mutant 3B that the ⁴AGP⁶ of 3B₁ and3B₂ were substituted by ⁴TAA⁶ was successfully constructed and expressed in BL21 bacteria.The result of Western Blot showed that the mutant 3B doesn't react with the monoclonal antibody3B4B1 contrary to the protein of rvOZK/93-08 3B,indicating that the epitope was deleted successfully.However after purification,concentration and emulsification,the serum of mice and guinea pigs immunized with the mutant 3B showed less differences with the rvOZK/93-08 3B tested by blocking ELISA.Presumably the reason was that the immunogenicity of the prokaryotic expression protein is different from that of the eukaryotic nucleus or the space barrier of the near epitopes have negative effects on blocking rate,indicating that the diagnostic methods of ELISA needs to be further improved still.In summary,the crucial amino-acid residues recognized by the monoclonal antibody 3B4B1were identified successfully,and a negative marker virus having similar ability replication with the parental virus was constructed successfully through the reverse genetics technology.The recombinant marker virus can be used for developing marker vaccine.