[Preparation and characterization of the polyclonal antibody against pig integrin beta6 subunit LBD as FMDV receptor].
OBJECTIVE
To induce the expression of FMDV receptor integrin beta6 subunit ligand-binding domain in E.coli and prepare the rabbit polyclonal antibody against it.


METHODS
The fragment coding beta6 ligand-binding domain was amplified by PCR and doubly digested with BamH Iand Xho I. Then it was cloned into expression vector pGEX-4T-1 to obtain recombinant plasmid pGEX-4T-1-beta6LBD. After pGEX-4T-1-beta6LBD was transformed into E.coli BL21(DE3) and induced by IPTG, the expression of fusion proteins was identified by SDS-PAGE, with inclusion body prepared and fusion protein purified. Then new Zealand rabbits were immunized to prepare polyclonal antibody against beta6LBD. GST-beta6LBD antiserum was obtained and the specificity of polyclonal antibody was detected by Western blot.


RESULTS
SDS-PAGE demonstrated that the fusion protein GST-beta6LBD was expressed with the expected molecular weight at 42 000. A single clear band of GST-beta6LBD fusion protein appeared in SDS-PAGE gel after purification. The titer of the polyclonal antibody was above 1:12 800 and it is of high specificity.


CONCLUSIONS
The successful preparation of rabbit anti pig beta6LBD polyclonal antibody with high affinity and specificity will lay a foundation for further research into the function of integrin beta6 in FMDV infection.
Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
Shan-Dian
SD
Gao
Hui-Yun
HY
Chang
Jun-Zheng
JZ
DU
Jing-Feng
JF
Wang
Jian-Hua
JH
Zhou
Jian-Yong
JY
Zhao
Qing-Ge
QG
Xie
D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D029544:Foot-and-Mouth Disease Virus / Q000378:metabolism* / Q000472:pathogenicity; D039641:Integrin beta Chains / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D008297:Male; D016133:Polymerase Chain Reaction; D021122:Protein Subunits / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011817:Rabbits; D011991:Receptors, Virus / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D013552:Swine
D000906:Antibodies; D039641:Integrin beta Chains; D021122:Protein Subunits; D011991:Receptors, Virus; C072170:integrin beta6