利用反向遗传学原理构建B、E型肉毒神经毒素基因型特异性靶片段

目的 利用反向遗传学原理构建B、E型肉毒神经毒素(BoNT)基因型特异性靶片段质粒、菌株.方法 自GenBank获取BoNT基因序列,利用DNAMAN、Lasergene、VectorNTI等多种生物信息学分析软件和BLAST等网上数据分析平台进行综合分析,以锚定BoNT/B与BoNT/E基因中的型特异性靶片段,分别设计合成5条和10条DNA短链,经重叠PCR扩增获得完整的靶片段,纯化后连接到pMD 18-T载体上,转化至大肠杆菌DH5α感受态细胞中,质粒提取后进行酶切和测序鉴定.结果 分析了全球已报道的60条BoNT基因序列(A、B、E、F型),于BoNT/B与BoNT/E基因中,分别锚定了215 bp和360bp的靶片段,并设计合成其特异的检测引物对,经酶切、测序确证,成功合成具有型特异性的BoNT/B与BoNT/E靶片段.结论 获得含有型特异性BoNT/B与BoNT/E靶片段的重组质粒pMD18-T-BoNT/B,pMD18-T-BoNT/E及其菌株.

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. CONCLUSION: The engineered plasmids and E.coli stains containing the genotype-specific target fragments of BoNT/B and BoNT/E have been constructed successfully.