[Establishment of the genotype-specific targets of botulinum neurotoxins types B and E based on reverse genetics].
OBJECTIVE
To establish the genotype-specific targets plasmids and engineered E.coli strains of botulinum neurotoxins (BoNT) types B and E based on reverse genetics.


METHODS
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.


RESULTS
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.


CONCLUSIONS
The engineered plasmids and E.coli stains containing the genotype-specific target fragments of BoNT/B and BoNT/E have been constructed successfully.
Li
Ying
Y
Department of Parasitology, School of Public Health and Tropical Medicine, NanFang Hospital Southern Medical University, Guangzhou, China. liying20920@hotmail.com
Yang
Chun-li
CL
Zhao
Ying
Y
Guo
Yu-qi
YQ
Liu
Chang-zheng
CZ
Zhou
Xiao-hong
XH