B型肉毒毒素重链C端基因的克隆与表达

目的:扩增B型肉毒毒素重链C端基因并将其在大肠杆菌中表达.方法:首先克隆B型肉毒毒素重链C端片段(BoNTB/Hc),经IPTG诱导,在大肠杆菌中进行天然序列的表达.构建原核表达载体pET32/Hc后进行融合蛋白的表达.对5′端引物进行修饰,最终进行N端修饰蛋白的表达.结果:扩增得到的BoNTB/Hc与已知序列同源性达99%,未得到天然序列的表达,获得了融合蛋白和N端修饰蛋白的表达.Western blot鉴定结果表明,融合蛋白和N端修饰蛋白都可以和特异性抗体发生反应.结论:获得了B型肉毒毒素重链C端基因的表达,为肉毒毒素相关研究奠定了基础.

Cloning and Expression of Hc Subunits from Clostridium Botulinum Type B

Objective:To clone and express Hc Subunits from Clostridium botulinum type B in E.coli.Methods:The carboxy-terminal end of the Hc containing the major determinants responsible for specific toxin was cloned and the natural Hc protein was expressed.In order to express the fusion protein,insertion of the Hc into prokaryotic expression vector pET32 was made.After the upstream prime was revised,the Hc N-terminal modified protein was achieved.Results:The identity of the cloning and Genebank sequences were up to 99%.The natural Hc protein was not obtained after being induced by IPTG,and then the fusion protein and modified proteins were harvested and identified by western blot.Conclusion: The successful cloning and expression of BoNTB/Hc are obtained,which is supposed to lay a foundation for the research of botulinum neurotoxins.