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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 type of A-superfamily conotoxin Bt14.10 from Conus betulinus in the sea area near the Xisha area in the South China Sea. The disulfide bond was identified by chemical method. Methods Genomic DNA of Conotrophic tuberosomatism was extracted. Based on the untranslated region and intron sequences, a synthetic primer was designed for PCR. The sequence of Bt14.10 was obtained. The linear peptide Bt14.10 was synthesized by Fmoc-solid phase method. The disulfide bond-containing folded product was obtained by air oxidation and the disulfide bond was determined by two-step folding method. As a result, a new conotoxin DNA sequence Bt14.10 was found, which encodes the mature peptide sequence CAHSVPGMHPCKCNNTC-NH2. The disulfide bond mode is “C1-C3, C2-C4”. Conclusions BT14.10 is a new type of superfamily of conotoxins with a type ¢ ô cysteine skeleton structure that is rare in the A superfamily conotoxins.