治疗用A型肉毒梭菌神经毒素全基因克隆及序列分析

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。

Cloning and Sequencing of Complete BoNT Gene of Clostridium botulinum Type A for Therapy

Objective To clone and sequence the complete BoNT gene of Hall strain of Clostridium botulinum type A for production of type A botulinum toxin for therapy in China,investigate its genetic characters and provide a basis for quality control of type A botulinum toxin.Methods Complete BoNT gene was amplified by PCR from the master and working seeds for production of type A botulinum toxin for therapy,and cloned into vector pGEM-T to construct recombinant plasmid pGEM-T-BoNT for sequencing.Results The cloned complete BoNT gene sequence of Hall strain,at a length of 3 891 bp,encoded 1 297 amino acids.The proportions of nucleotides A,G,T and C were 40.58%,16.17%,33.13% and 10.13%,while the GC and AT contents were 26.29% and 73.71%,respectively.A nonsense mutation(G→A at site 3591) was observed between master and working seeds,resulting no change of deduced amino acids.The homologies of nucleotides and amino acids of both master and working seeds to those of standard Hall strain in GenBank were 99.99% and 100% respectively.Conclusion The BoNT gene of Hall strain of Clostridium botulinum type A was highly stable in genetic during long-term storage in laboratory and subculture for production.