Cloning Of The Nonstructural Protein Gene 3ABC Of Foot-and-Mouth Disease Virus And Expression Of The 3AB Gene In Escherichia Coli

Foot-and-mouth disease(FMD) is a highly contagious disease of cloven-hoofed animals, whose causative agent,foot-and-mouth disease virus(FMDV),is a member of the Aphthovirus genus within the Picornaviridae. FMDV usually causes persistent infection or inapparent infection that can also occur in vaccinated animals following contact with the virus,which will influence the diagnostic techniques and the preventive measures that need to be taken. Therefore, how to determine inapparent infection and differentiate between infected and vaccinated animals is required for FMD prevention and control.In this paper,first strand cDNA of 3ABC gene was synthesized using template RNA extracted from cells infected with FMDV.The complete 3ABC gene about ISOObp was amplified by PCR and ligated into pGEM-T Easy vector.After transforming E.coli DH5 a ,ampicillin resistant colonies were isolated and plasmid DNA was prepared and analyzed by restriction analysis and PCR.Presence of the full length 3ABC gene was verified by nucleotide sequence analysis and the plasmid containing the expected sequence was named as pGEM-3ABC.Comparing the aquired sequence of 3ABC with that of reference strains,the homology is more than 99 percent .The pGEM-3ABC was digested with Sal I and Bgl II and Ligated into Xho I and Bgl II -digested expression vector pTriEx-4 Neo.lt was identified by restriction analysis and PCR and sequencing that this fragment had a 17bp deletion hi the nucleotide sequence 708bp of 3ABC gene,which happened to form a terminator codon behind 3AB gene,but it contained the complete open reading frame(ORF)of 3AB gene.Positive clones were selected and induced with lmmol/L Isopropyl-D-galactoside(IPTG),bacteria were detected by SDS-PAGE and Western blotting after properly treated.The results showed that the 3AB gene expressed successfully in E.coli and 33.5ku fusion protein can be recognized by the positive bovine serum of FMDV.The amount of target protein is over 26% of the total bacteria protein by gel thin layer scanning analysis.