海南产大尉芋螺的新型O-超家族芋螺毒素基因克隆

Cloning of novel O-superfamily conotoxin of Conus capitaneus collected from Hainan

目的从海南产大尉芋螺毒液中分离克隆新型O-超家族芋螺毒素基因,为大尉芋螺毒素药物的研究与开发提供源头化合物。方法采用快速扩增cDNA 3’-末端(3’-RACE,Rapid Amplification of cDNA Ends)的方法,对我国海南产大尉芋螺(Conus capitaneus Linnaeus)的毒素cDNA进行分析,克隆测序所得基因片段,再进行序列比较,以及O-超家族芋螺毒素基因的遗传变异分析。结果发现了1种新的O-超家族芋螺毒素cDNA序列CaHr91N,编码长度为77个氨基酸(aa)的前体蛋白CaHr91P,具有典型的芋螺毒素前体结构特征,即由21aa的信号肽、22aa的前肽与34aa的成熟肽三部分组成。毒素区的成熟肽CaHr91M序列为ECREQSQGCTNTSPPCCSGLRCSGQSQGGVCISN,具有O-超家族芋螺毒素保守的半胱氨酸框架C-C-CC-C-C.同时比较分析了CaHr91P与其它同源性的O-超家族芋螺毒素的相似性,以及大尉芋螺毒素的研究状况。结论CaHr91P新芋螺毒素基因的发现,及其序列的阐明,为进一步研究其生物活性和应用奠定了基础,同时为更多大尉芋螺毒素的发现和研究利用积累了经验。

Objective To isolate and clone novel O-superfamily conotoxin genes of Conus capitaneus Linnaeus collected from Hainan, which would provide the initial drug leads to investigate and develop Conus capitaneus conotoxins. Methods 3＇-RACE （Rapid Amplification of cDNA Ends） was used for cloning the novel O-superfamily conotoxins. The specific amplified cDNA fragments were sequenced and analyzed, as well as the genetic diversity of the O-superfamily conotoxins. Results The full-length cDNA （CaHr91N） of a new O-superfamily conotoxin （CaHr91） was cloned and sequenced from Conus capitaneus Linnaeus. The novel conotoxin precursor CaHr91P with 77 amino acids （aa） encoded by the cDNA consists of three typical regions of signal with 21aa, pro-peptide with 22aa and mature peptide with 34aa. Predicted sequence of the toxin region CaHr91M is ＂ECREQSQGC TNTSPPCC SGLRC SGQSQGGVC ISN＂ with a common O-superfamily cysteine pattern C-C-CC-C-C. Percent identities between CaHr91P and other published homologue O-superfamily sequences were compared systematically, as well as research status on conopeptides from C. capitaneus. Conlusion The elucidated cDNA of the novel CaHr91P conotoxin will be the basis for a better understanding of its bioactivity and application, as well as finding more novel conotoxins from C. capitaneus.