*4.5. Detection via AflR Gene Analysis and Transformation*

4.5.1. Cloning, Sequencing, and *AflR* Gene Transformation

The PCR product (Section 2.4) was excised from the gel and purified using a QIA quick gel extraction kit (Qiagen Inc., Berlin, Germany). Purified DNA was ligated into the pGEM-T vector (Promega Co., New York, NY, USA). Recombinant plasmids were directly sequenced using an automated sequencer (Macrogene Company, Seoul, Korea), with a universal vector primer. DNA homology searches were carried out using the NCB1 databases and the BLAST network service. *EcoR*I and *No*tI restriction enzymes were used for gene release and insertion into the pPROEX HTa expression vector (Life Technologies, New York, NY, USA). The recombinant plasmid was transformed into competent *E. coli* (BL21) cells and recombinant protein was recovered as previously described [29].

#### 4.5.2. Molecular Size Determination of *AflR* Recombinant Protein

The recombinant protein was separated on 12% SDS PAGE and molecular size determined using a standard low range protein marker (BioRad, Hercules, CA, USA). Gel preparation, staining, and destaining were carried out following Laemmli [30].

#### 4.5.3. Epitope Prediction and Antigenic Determination

B-cell epitope prediction analysis was performed following Kolaskar and Tongaonkar [16] to examine the epitope in different antigenic determinants.
