Expression and serological application of recombinant epitope-repeat protein carrying an immunodominant epitope of Newcastle disease virus nucleoprotein

Gaikwad, Satish S.; Lee, Hyun-Jeong; Kim, Ji-Ye; Choi, Kang-Seuk

The construct encoding ERP on NDV NP was designed as shown in Fig. 1A . The codon optimized gene (486 bp) was designed to have 8 repeats of 16 amino acids (ETQFLDLM-RAVANSMR) mimicking C-terminal IDE (aa 444-459) on NDV NP. rERP gene insert was excised from the plasmid after digestion with EcoRI and HindIII enzymes. Resulting fragment (486 bp) was subcloned into the pFastBac HT b vector using above mentioned restriction sites. Recombinant baculovirus containing the recombined ERP gene insert (bac-rERP) was generated by transposition of recombinant vector into the SF9 cells by transfection. The codon optimized gene was commercially synthesized from GeneArt (Regensburg, Germany) and subcloned into baculovirus transfer vector. Alignment of obtained nucleotide and deduced amino acid sequences of the rERP gene insert revealed 100% match with the designed construct (data not shown). I-TASSER server predicted 5 models, all of which showed optimal spacing in epitopes separated by linkers (Fig. 1B) . Graphic visualization of tertiary structure of top predicted models for rERP protein suggests that IDE reactive antibodies would be freely accessible to epitopes separated by tetra-glycine linkers in tertiary dimensional space.