[Prokaryotic expression, protein purification and reactivity analysis of the VP1 and its C terminus of a South African isolate of FMDV serotype SAT2].
OBJECTIVE
To induce the expression of structure protein VP1 and its C terminus of foot-and-mouth disease virus (FMDV) serotype SAT2 in E.coli and analyze their reactivities with FMDV positive antiserum.


METHODS
The plasmid pGEM-SAT2P1 carrying the VP1 coding region of FMDV serotype SAT2 isolated from South African was used as template for RT-PCR to get the coding fragment of VP1 and its C terminus. The fragments were then cloned into expression vector pET-30a(+) to get recombinant plasmids pET-SAT2VP1 and pET-SAT2VP1C. The recombinant plasmids were transformed into E.coli BL21(DE3)pLysS and induced by IPTG to express VP1 and its C terminus protein. The expressed VP1 and its C terminus were then purified by Ni-NTA His Bind Resin affinity chromatography and analyzed by Western blot. New Zealand rabbits were immunized to prepare polyclonal antibodies against VP1 and VP1C. The antisera were obtained and polyclonal antibody was characterized by ELISA.


RESULTS
SDS-PAGE demonstrated that VP1and its C terminus expressed in the E.coli transformants had a molecular weight of 33000 and 19000 and contained in the inclusion body. Purified VP1 and its C terminus was obtained by Ni-NTA His Bind Resin affinity chromatography and a single clear band appeared in the SDS-PAGE gel. Western blot analysis showed that the purified VP1 and VP1 C terminus could react with bovine antiserum against the same serotype FMDV without cross-reactivity with the negative bovine serum.


CONCLUSIONS
Rabbit polyclonal antibodies against VP1 and VP1C were successfully prepared, the titers of which were above 1:12800 and had obvious specificity.
Gao
Shan-dian
SD
Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, National FMD Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China. gaoshandian1@126.com
Chang
Hui-yun
HY
Du
Jun-zheng
JZ
Shao
Jun-jun
JJ
Cong
Guo-zheng
GZ
Lin
Tong
T
Han
Xue-qing
XQ
Xie
Qing-ge
QG
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D002417:Cattle; D002418:Cattle Diseases / Q000276:immunology* / Q000821:virology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005536:Foot-and-Mouth Disease / Q000276:immunology* / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000145:classification / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D015870:Gene Expression; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013019:South Africa
D000914:Antibodies, Viral; D036022:Capsid Proteins; C442501:VP1 protein, Foot-and-mouth disease virus