Establishment of the genotype-specific targets of botulinum neurotoxins types B and E based on reverse genetics / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To establish the genotype-specific targets plasmids and engineered E.coli strains of botulinum neurotoxins (BoNT) types B and E based on reverse genetics.</p><p><b>METHODS</b>The gene sequences of BoNT were obtained from GenBank and analyzed using DNAMAN, Lasergene, Vector NTI and BLAST. Two target fragments of BoNT/B and BoNT/E were anchored and then synthesized as 5 and 10 short DNA single strands, respectively. The full-length target sequences were amplified by overlapping PCR and subcloned into pMD 18-T vector, and the recombinant plasmids were identified by restriction enzyme digestion and sequencing.</p><p><b>RESULTS</b>Sixty full-length sequences of 4 types of BoNT, namely types A, B, E, and F, were available in GenBank. Two target fragments, BoNT/B of 215 bp and BoNT/E of 360 bp, and their specific primer pairs were anchored after sequence analysis. pMD 18-T-BoNT/B and pMD 18-T-BoNT/E containing these two target sequences were confirmed.</p><p><b>CONCLUSION</b>The engineered plasmids and E.coli stains containing the genotype-specific target fragments of BoNT/B and BoNT/E have been constructed successfully.</p>