Establishment of the genotype-specific targets of botulinum neurotoxins types B and E based on reverse genetics.
- Author:
Ying LI
1
;
Chun-li YANG
;
Ying ZHAO
;
Yu-qi GUO
;
Chang-zheng LIU
;
Xiao-hong ZHOU
Author Information
- Publication Type:Journal Article
- MeSH: Base Sequence; Botulinum Toxins; genetics; Botulinum Toxins, Type A; Clostridium botulinum; genetics; isolation & purification; Gene Targeting; Genotype; Molecular Sequence Data; Polymerase Chain Reaction; methods; Sequence Analysis, DNA
- From: Journal of Southern Medical University 2008;28(8):1382-1386
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo establish the genotype-specific targets plasmids and engineered E.coli strains of botulinum neurotoxins (BoNT) types B and E based on reverse genetics.
METHODSThe 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.
RESULTSSixty 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.
CONCLUSIONThe engineered plasmids and E.coli stains containing the genotype-specific target fragments of BoNT/B and BoNT/E have been constructed successfully.
