[Secreted expression of nonstructural protein gene 3ABC of foot-and-mouth disease virus in Sf9 cells and activity analysis].
Entire 3ABC sequence of FMDV containing a 6 x his tag coding sequence at the N-terminal was obtained through PCR amplification using a pair of specific primers, subcloned into shuttle plasmid of pMelBac-B with a melittin secretion signal sequence and finally constructed recombinant plasmid of pMel-3ABC. After co-transfected the recombinant plasmid and linearized Bac-N-Blue DNA into Sf9 insect cell under intermediary agent of the Cellfectin, the result showed that we have already acquired recombinant baculovirus by screen of plaque assay and identification of PCR. Though the recombinant baculovirus infecting the Sf9 cells again, experiments indicated that 3ABC gene could express in insect cells and the expressed protein was secreted in the supernatant of Sf9 cell culture possessing favourable biological activities detected by adopting two methods of SDS-PAGE and Western blot. The result verified that the protein could respond with sera derived from FMDV infected animals, but have no responsibility with sera derived from health animals and vaccinated animals detected by indirect ELISA using antigen of expressed protein after purification with Ni-NTA his bind resin. Therefore, this study has established a solid foundation for establishing an effective diagnosis method to discriminating the FMDV infected animals from vaccinated animals.
Ma
Jiang-Tao
JT
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute Chinese Academy of Agricultural Science, Lanzhou 730046, China.
Lu
Zeng-Jun
ZJ
Cao
Yi-Mei
YM
Guo
Hui-Chen
HC
Guo
Jian-Hong
JH
Zhu
Xiu-Mei
XM
Yang
Xiao-pu
XP
Liu
Zai-Xin
ZX
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D017077:Culture Media, Conditioned / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005536:Foot-and-Mouth Disease / Q000276:immunology / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D007106:Immune Sera / Q000276:immunology; D010957:Plasmids / Q000235:genetics*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012756:Sheep; D018411:Spodoptera; D013552:Swine; D014162:Transfection / Q000379:methods; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
C109754:3ABC protein, virus; D000956:Antigens, Viral; D017077:Culture Media, Conditioned; D007106:Immune Sera; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins