[Cloning and expression of 3C protease gene from foot-and-mouth disease virus in insect cell].
The 3C protease from foot-and-mouth disease virus (FMDV 3Cpro) is critical for viral pathogenesis, has vital roles in both processing of the polyprotein precursor and RNA replication, and is a potential anti-viral drug target. In the study, 3C gene of FMDV from serotype Asia I was obtained through Polymerase Chain Reaction (PCR), and subcloned into baculovirus transfer vector pMelBac-B. The recombinant transfer plasmid and linearized baculovirus DNA were co-transfected into sf9 insect cell, and the recombinant baculovirus were screened by plaque cloning and PCR identification. After amplification of recombinant baculovirus on cell passages, the recombinant virus were seeded on sf9 cell with 10 multiplicity of infection (MOI), and cells were harvested 72 hours after infection. The expressing product of 3C gene in insect cells was detected by Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and Western blot. The result demonstrated that the 3C gene was successfully expressed in insect cells. The product was a 23 kDa protein and could be recognized by anti-FMDV serum in western blot. The results provide a basis for research of the assembly of FMDV empty capsids in vitro and the design of antivirus drug.
Cao
Yimei
Y
Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Science, State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou, 730046, China. caoym518@126.com
Lu
Zengjun
Z
Sun
Pu
P
Sun
Jiachuan
J
Liu
Zaixin
Z
D000086042:3C Viral Proteases; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018411:Spodoptera / Q000166:cytology*; D014764:Viral Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism
D004274:DNA, Recombinant; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases