A型肉毒毒素保护性抗原基因的克隆及其结构分析

Cloning and Sequencing of Protective Fragment (Hc) of Botulinum Neurotoxin Serotype A(BoNTa)

以文献报道的 A型肉毒神经毒素基因全序列为标准 ,设计并合成一对引物 ,从肉毒梭菌中扩增含保护性抗原表位的肉毒毒素重链 C端 (Bo NTa Hc)基因片段 ,并将扩增产物进行限制性内切酶消化 ,与相同酶处理的克隆测序载体 p Bluescript KS( +)在体外连接 ,构建测序重组质粒 ,进行测序和基因结构分析 .结果 :PCR扩增获得了产物为 12 75 bp大小的 DNA片段 ,测序结果与 DNA数据库对照检索分析证明 ,此基因片段与 Genbank中的 Bo NTa Hc基因的同源性为 99%,可以认为克隆的基因即为 Bo NTa

The C terminal half of the heavy chain gene of botulinum neurotoxin serotype A (BoNTaHc) was amplified and cloned into a sequencing plasmid pBluescript KS(Ⅱ +). Cloned gene was sequenced and analyzed.A pair primers based on BoNTa complete sequence from Genbank and performed PCR amplification was designed. PCR product digested by restrictive endonclease was ligated into pBluescript. Cloned gene was analyzed. Result: A 1 275 bp DNA fragment was amplified. Recombinant of pBlue BoNTaHc was constructed. Sequence analysis proved that the cloned gene was BoNTaHc gene. The successful cloning of BoNTaHc gene is the key to express in E.coli and further study on vaccine.