Epitope Maping Of Odontoglossum Ringspot Virus Coat Protein And Application For Purification Of Specific Antibody

Odontoglossum ringspot virus?ORSV?,one typical member of the Tobamovirus genus of plant virus,could damage the value of orchid in economy and ornamentary aspects severely.The high quarantine requirements of ORSV in the international market lead to a large need of the test kits for detection of ORSV.However,due to the obvious serological cross-reactivity between ORSV and other Tobamoviruses,the low accuracy of commercial serological test kits can't meet the requirement of users nowadays.Using anti-ORSV polyclonal antibody as the target protein,peptides which could bind with antibody were selected from the phage display 7 peptide library.5147 peptides gene were sequenced by Miseq and 4681 phage clones were effective.Further analysis showed that the amino acid sequence of 58 clones are highly homologous with ORSV coat protein,mainly in 1?35_aa,43?68_aa,107?153_aa.According to the conservation in amino acid sequence among ORSV,TMV and ToMV,we concluded that preliminary specific region epitope of ORSV were mapped to:₂SYTITDPSKLAYLSSAWADPNSLINLCTN₃₀,₅₀ADVWQPVPTLTSRFPAGAGYFR₇₁ and ₁₄₁MYNQVSFETMSGLTWTSS₁₅₈.Fifteen fusion proteins carrying the Trx.tag and the peptides of ORSV CP in the C-terminal were expressed in E.coli.The affinity between fusion proteins and ORSV polyclonal antibody was detected by indirect ELISA.The results showed that five fusion proteins with peptides from ORSV CP 1-30_aa region,four fusion proteins with peptide from ORSV 56-75_aa region,had high affinity with ORSV polyclonal antibody.These results provided further evidence that the specific epitope located at:₂SYTITDPSKLAYLSSAWADPNSLINL₂₇,₅₆VPTLTSRFPAGAGYFR₇₁.Three peptides,ORSV1(₂SYTITDPSKLAY₁₃),ORSV2(₁₅SSAWADPNSLINL₂₇)and ORSV3(₅₈TLTSRFPAGAGYFR₇₁)were synthesized by Fmoc chemistry method.Three affinity chromatograph gel were product by conjugated the peptides to CNBr activated Sepharose FF,respectively.Three specific antibodies for epitope,Anti-ORSV1,Anti-ORSV2,Anti-ORSV3,were purified using the affinity chromatograph gel.The Anti-ORSV3 has high specificity than the polyclonal antibody,and is suitable for replacement of polyclonal antibody in the development of test kits.