Aim: To clone and express the ligand binding domain (LBD) cDNA of porcine integrin beta3 as foot-and-mouth disease virus (FMDV) receptor and prepare its polyclonal antibody.
Methods: The LBD cDNA of porcine beta3 was obtained from the lung tissue of pig infected with FMDV by RT-PCR, and the recombinant plasmid pGEM/beta3LBD was constructed. After digested with BamH I/Xho I, the beta3LBD fragment was subcloned into prokaryotic expression vector pGEX 4T-1. The recombinant expression plasmid pGEX/beta3LBD was constructed and transformed into E.coli BL21(DE3). The recombinant porcine beta3LBD protein was expressed after IPTG induction and purified from total protein of BL21(DE3). The rabbits from New Zealand were immunized with the purified fusion protein to prepare polyclonal antibody, which was identified by Western blot and ELISA.
Results: The 507 bp cDNA of porcine beta3LBD encoded a polypeptide of 169 amino acids. The similarity of nucleotide sequence beta3LBD between pigs and cattle, human being, chimpanzees, rhesus monkeys, horses, dogs, Norway rats, mice, chickens was 90.3%, 92.3%, 92.1%, 91.3%, 90.5%, 90.3%, 87.8%, 85.2%, 79.5%, respectively. The beta3LBD gene of mammals exhibited high sequence homology. The recombinant beta3LBD protein was expressed efficiently as inclusion body after IPTG induction and was approximately 44000. The titer of the polyclonal antibody against the purified beta3LBD protein was about 1:12 800 by ELISA.
Conclusion: The gene cloning and expression of beta3LBD and the preparation of its polyclonal antibody lay a foundation for further research into the interaction of FMDV with beta3 subunit of porcine integrin.