广西产眼镜王蛇毒α-神经毒素基因的分子克隆及序列分析  

Cloning and Sequence Analysis of the cDNA Encoding of α-Neurotoxin from Guangxi King Cobra (Ophiophagus hannah)

作　　者：李其斌[1] 黄光武[1] 赵晓琴[1] 渡庆次贺博 金城记代彦 中村真理子 小杉忠诚[1,2] 

机构地区：[1]广西医科大学第一附属医院急诊科蛇伤救治与蛇毒临床应用研究中心 [2]日本琉球大学医学部生理学科

出　　处：《广西科学》2002年第3期202-206,共5页Guangxi Sciences

摘　　要：为获得眼镜王蛇 (Ophiophagus hannah ,简称 Oh)蛇毒 α-神经毒素 (α- NT)的基因序列 ,依据眼镜蛇科不同毒蛇种类来源的 α- NT基因有较高的同源性 ,设计 1对上下游引物 ,为克服引物带来模糊扩增 ,在蛋白编码部分再设计 1对上下游特异引物 ,用 Nacleospin RNA Kit法从 3条活眼镜王蛇毒腺中提取 m RNA,以 3′端引物合成的 c DNA作为模板进行 PCR扩增反应 ,测定产物的核苷酸序列 ,得到全长 4 74 bp的眼镜王蛇 c DNA基因核苷酸序列。该核苷酸序列的信号肽与眼镜蛇树属 Pseudonnaja textilis(Pt)、海蛇 L aticauda semif asciata (L s) 10 0 %同源 ,与眼镜蛇南洋亚种 N aja sputatrix (Ns)、银环蛇 (Bungarusmulticinctus) (Bm) 96 .8%同源 ;蛋白密码部分有83.3%与 Ns、79.2 %与 Pt、76 .4 %与 L s、74 .1%与 Bm同源。信号肽后紧接着的 72个氨基酸有 90 .3%与已发现的眼镜王蛇毒长链α- NT Toxin a同源 ,大约有 73.6 %与 Toxin b、6 9.7%与 Oh- 4、6 6 .7%与 Oh- 5、5 6 .9%与 Oh-6 A和 6 B同源 ,并与 α-银环蛇毒素 5 4 .2 %同源。说明新发现的眼镜王蛇 c DNA是一条长链 α- NT基因。To obtain the cDNA sequence of α neurotoxin (α NT)from Guangxi king cobra(Ophiophagus hannah,Oh),the sense primer was designed from the 5′ conserved regions with the start codon ATG,and the antisense primer was used as the d(T) for amplification of cDNA 3′end (RACE PCR) Two primers were designed from the 5′,3′ coding regions for overcoming the mistake amplification by the primer The venom gland RNA was extracted from three Oh snakes with the isolation kit The extracted RNA was reversely transcribed into cDNA with the oligo(dT) antisense primer The nucleotides sequence was analyzed on the ABI PRISM 310 automatic DNA sequencer The nucleotide sequence of 474 base pairs consisting of a 5′ untranslated region (6bp),signal peptide with ATG (63 bp), protein coding region (216 bp) and 3′ untranslated region (186 bp) with a TGA termination codon was obtained The comparison of the cDNA of long chain α NT between Oh snakes and other snakes reveals that the nucleotide sequence is much higher corresponding to the signal peptide than the mature protein coding regions which is 100% identical to Pseudonnaja textilist (Pt) and Laticauda semifasciata (Ls),96 8% to Naja sputatrix(Ns) and Bungarus multicinctus(Bm) The conservation of mature protein regions are about 83 3% to Ns,79 2% to Pt,76 4% to Ls and 74 1% to Bm snakes It is found that the conservation of Oh α NT from cDNA 90 3% is identical to Oh α NT Toxin a,about 73 6% to Oh Toxin b,69 7% to Oh 4,66 7% to Oh 5,56 9% to Oh 6A and Oh 6B;and 54 2% to α bungarotoxin The result indicates that the Oh cDNA is a long chain α NT gene

关 键 词：蛇毒α-神经毒素 分子克隆 眼镜王蛇 基因序列 

分 类 号：R996.3[医药卫生—毒理学]