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  

Establishment of the genotype-specific targets of botulinum neurotoxins types B and E based on reverse genetics

作　　者：李莹[1] 杨春莉[2] 赵莹[1] 郭瑜琪[1] 刘昌政[1] 周晓红[1] 

机构地区：[1]南方医科大学公共卫生与热带医学学院病原生物学系,广东广州510515 [2]南方医科大学南方医院检验科,广东广州510515

出　　处：《南方医科大学学报》2008年第8期1382-1386,1390,共6页Journal of Southern Medical University

基　　金：广东省重大专项子课题(2006B33761002)

摘　　要：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、Vector NTI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pMD18-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215bp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。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.

关 键 词：反向遗传学 肉毒神经毒素 克隆 

分 类 号：Q78[生物学—分子生物学]