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目的对我国治疗用A型肉毒毒素生产用菌株Hall株神经毒素(BoNT)全基因进行克隆及序列分析,了解其遗传特性,为该制剂的质量控制提供依据。方法取生产用主代种子、工作种子,通过PCR扩增BoNT全基因片段,将其克隆至载体pGEM-T中,构建重组克隆质粒pGEM-T-BoNT,对克隆的BoNT基因进行序列测定与分析。结果测得的Hall株BoNT全基因序列3891bp,共编码1297个氨基酸。4种核苷酸的比例分别为:A:40.58%,G:16.17%,T:33.13%,C:10.13%;GC含量为26.29%,AT含量为73.71%。主代种子、工作种子核苷酸序列3591位的G变成A,为无义突变,未导致其推导的氨基酸的改变。序列测定结果与GenBank中登录的Hall标准株进行比较,主代种子、工作种子核苷酸和氨基酸序列的同源性分别为99.99%和100%。结论 A型肉毒梭菌Hall株在实验室长期的保存和生产传代过程中,BoNT基因遗传特性非常稳定。
Objective To clone and sequence the full-length gene of Neurotoxin (BoNT) from Hall strain of botulinum toxin type A production in our country, and to understand its genetic characteristics and provide the basis for the quality control of this preparation. Methods The main generation seeds and working seeds were taken for production. The whole BoNT gene fragment was amplified by PCR and cloned into vector pGEM-T. The recombinant plasmid pGEM-T-BoNT was constructed and sequenced and analyzed. . Results The total length of BoNT gene of Hall strain was 3891bp, encoding a total of 1297 amino acids. The proportions of the four nucleotides were: A: 40.58%, G: 16.17%, T: 33.13%, C: 10.13%; GC content was 26.29% and AT content was 73.71%. The main seed, working seed nucleotide sequence of 3591 G becomes A, nonsense mutation, did not lead to its deduced amino acid changes. The results of sequence analysis were compared with those of Hall standard strains registered in GenBank. The homologies of nucleotide and amino acid sequences of main seeds and working seeds were 99.99% and 100% respectively. Conclusion The Clostridium botulinum type Hall strain has a very stable genetic characteristics of BoNT gene during long-term preservation and production passaging in the laboratory.