A pair of specific primers was designed from the 2C gene sequence of foot-and-mouth disease virus(FMDV)for amplification of a fragment including 174bp of the 5'-end and 279bp of the 3'-end of the2C gene,which ...A pair of specific primers was designed from the 2C gene sequence of foot-and-mouth disease virus(FMDV)for amplification of a fragment including 174bp of the 5'-end and 279bp of the 3'-end of the2C gene,which encoded an abundance of known B-cell epitopes of the protein.The amplified fragment was inserted into pET-30a plasmid(Novagen)via two unique endonuclease restriction sites,Nco I and Sal I.Sequencing confirmed that the open reading frame of interest was correctly inserted into the positive recombinant plasmid.The positive plasmid was transformed into the host bacteria BL21(DE3)pLys for protein expression.After induction by IPTG at 37℃for 5 hours,the expressed product was analyzed by SDSPAGE and Western blotting,confirming successful expression.The product is a 23kDa fusion protein and was shown to react with sera derived from FMDV-infected animals.This approach provides an useful antigen for establishing an enzyme-linked immunoelectro-transfer blot assay(EITB)diagnostic method,useful for differentiating FMDV-infected animals from those that been vaccinated.展开更多
基金supported by the National Key Technolo-gies R&D Program(2006BAD06A12)the Key Project of Chinese National Programs for Fundamental Research and Development(2005CB523201)
文摘A pair of specific primers was designed from the 2C gene sequence of foot-and-mouth disease virus(FMDV)for amplification of a fragment including 174bp of the 5'-end and 279bp of the 3'-end of the2C gene,which encoded an abundance of known B-cell epitopes of the protein.The amplified fragment was inserted into pET-30a plasmid(Novagen)via two unique endonuclease restriction sites,Nco I and Sal I.Sequencing confirmed that the open reading frame of interest was correctly inserted into the positive recombinant plasmid.The positive plasmid was transformed into the host bacteria BL21(DE3)pLys for protein expression.After induction by IPTG at 37℃for 5 hours,the expressed product was analyzed by SDSPAGE and Western blotting,confirming successful expression.The product is a 23kDa fusion protein and was shown to react with sera derived from FMDV-infected animals.This approach provides an useful antigen for establishing an enzyme-linked immunoelectro-transfer blot assay(EITB)diagnostic method,useful for differentiating FMDV-infected animals from those that been vaccinated.
