新A超家族芋螺毒素Bt14.10的克隆及合成研究

Cloning and synthesis of a new A-superfamily conotoxin Bt14. 10

目的从我国南海西沙附近海域的桶形芋螺(Conus betulinus)中克隆出新型A-超家族芋螺毒素Bt14.10,并利用化学方法合成该毒素,鉴定其二硫键连接方式。方法提取桶形芋螺毒腺管基因组DNA,基于非翻译区及内含子序列设计合成引物进行PCR,获得Bt14.10的序列。采用Fmoc-固相法合成线性肽Bt14.10,通过空气氧化折叠获得含二硫键的折叠产物,利用两步折叠法测定其二硫键连接方式。结果发现一种新的A超家族芋螺毒素DNA序列Bt14.10,其编码的成熟肽序列为CAHSVPGMHPCKCNNTC-NH2,二硫键连接方式为"C1-C3,C2-C4"。结论BT14.10是一种新型A超家族芋螺毒素,具有A超家族芋螺毒素较为少见的ⅪⅤ型半胱氨酸骨架结构。

Objective To clone a new conotoxin Bt14.10 from Conus betulinus derived from the South China Sea, synthesize the peptide , and to determine linkage of its disulfide bridges .Methods The genomic DNA was extracted from C.betulinus venom duct while the Bt14.10 sequence was cloned using primers designed based on the untranslated region and intron.The peptide was then synthesized using solid-phase method and folded into the target product whose disulfide bridge connection was further determined by two-step oxidative folding .Results A novel conotoxin designated as Bt 14.10 （CAHSVPGMHPCKCNNTC-NH2） was obtained,the disulfide connectivity of which was ＆quot;C1-C3,C2-C4＆quot;.Conclusion Bt14.10 is a new A-superfamily conotoxin and has a distinct loop spacing pattern between cysteines in A-superfamily conotoxins.