Mapping Of Antigenic Epitopes And Constructing Of Single Chain Antibody On Soybean Mosaic Virus Coat Protein

Soybean(Glycine max),originated from China,is an important source of protein and fat in the world,and also occupies a prominent position in the national economy and daily life in China.Soybean mosaic disease is a major disease,caused by the pathogen of Soybean mosaic virus(SMV).In this study,we attempted to explore apossible way of anti-SMV genetic breeding of soybean using “plantibody”technology.To obtain the anti-SMVplantibody,firstly,the gene encoding the Coat Protein(CP)of SMV was expressed in prokaryotic cell.Then BABL/c mice were immunized with the recombinant CP,thespleen lymphocytes were separated and fused with Sp2/0cells.Through screening,10 strains of hybridoma cell linessecreting monoclonal antibodies anti-SMV were obtained: 2D3,4F9,4G12,6B10,5D2,2A8,3B8,6E7,2C1,1C2.The results of virus neutralization tests indicated that the 10 cell linesshowed different activities in neutralization.Among them,cell lines of 5D2,2A8,3B8,6E7,2C1 and 1C2 showed strong neutralizing activity;however,2D3,4F9,4G12 and 6B10 were less strong than the others.According to these results,all of the antibodies had the potential abilities of anti-SMV.The genes coding SMV CP could be cloned and transformed to plants to achieve their disease resistance in the form of plantibodies.Further experiments in mapping the epitopes on CP were carried on in order to select "plantibody" candidates in 10 monoclonal antibodies.Total of 5 antigenic epitopes were located.The epitopes CP-B5,CP-B6 and CP-B5+B6 located at the center of CP protein were very conservative among different strains of SMV,and highly conserved in potyvirus.Therefore,the epitope region is a suitable target for genetic breeding against potyvirus.Meanwhile,the encoding genes of monoclonal antibodies4G12,6B10,2D3 and 4F9 recognized this epitope region could becandidate genes for anti-SMV breeding.After identification,4 strains of monoclonal antibody 2D3,4F9,4G12 and 6B10 could at least be recognized 4 members of potyvirus,whichwere SMV,Potato virus Y(PVY),Onion yellow dwarf virus(OYDV)and Clover yellow vein virus(CLYVV).These broad-spectrum antibodies smight provide the materials in detection of potyvirus.OYDV was identified as the pathogen of mosaic disease on a local crop shallot by 4G12.Monoclonal antibody 2D3,4F9,4G12 and 6B10 could be used as broad-spectrum resistance genes against these 4 species of viruses.The variable region sequences of light and heavy chains of broad-spectrum monoclonal antibodies(2D3 and 4F9)were cloned.After connection by linker,single chain antibody 2D3-scfv and 4G12-scfv were constructed and expressed in Escherichia coli.Activity analysis showed that 4G12-scfv could recognize the CP protein of SMV,but the affinity of recombinant 2D3-scfv and 4G12-scfv decreased significantly,and there was nonspecific binding.The bindingability to 3 other viruses(PVY,OYDV,CLYVV)is very weak than to SMV,and their affinity was less than the 1/10 of the monoclonal antibody.In conclusion,10 strainmonoclonal antibodies with neutralizing activity anti-SMV were obtained in this study.By fine mapping of antigenic epitopes of SMV CP protein,3 highly conservative epitopes were found in different SMV strains and members of potyvirus.Monoclonal antibody 2D3,6B10,4F9 and 4G12 could be used as broadspectrum resistance genes to SMV,PVY,CLYVV and OYDV,in which the light and heavy chain variable region sequence of 2D3,4G12 were cloned.This study may provide a new target and broad-spectrum resistance genes for the molecular genetic breeding for resistance to SMV and potyvirus.