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李其斌,黄光武,赵晓琴,渡庆次贺博,金城记代彦,中村真理子,小杉忠诚.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[J].广西科学,2002,9(3):202-206. [点击复制]
- Li Qibin,Huang Guangwu,Zhao Xiaoqin,Y. Togesi,K. Kinjoh,M. Nakamura,T. Kosugi.Cloning and Sequence Analysis of the cDNA Encoding of α-Neurotoxin from Guangxi King Cobra (Ophiophagus hannah)[J].Guangxi Sciences,2002,9(3):202-206. [点击复制]
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| 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 |
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李其斌, 黄光武, 赵晓琴, 渡庆次贺博, 金城记代彦, 中村真理子, 小杉忠诚
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| (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, 南宁市滨湖路6号 530021) |
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| 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mRNA,以3'端引物合成的cDNA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474bp的眼镜王蛇cDNA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Laticauda semif asciata(Ls)100%同源,与眼镜蛇南洋亚种Naja sputatrix(Ns)、银环蛇(Bungarus multicinctus)(Bm)96.8%同源;蛋白密码部分有83.3%与Ns、79.2%与Pt、76.4%与Ls、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、69.7%与Oh-4、66.7%与Oh-5、56.9%与Oh-6A和6B同源,并与α-银环蛇毒素54.2%同源。说明新发现的眼镜王蛇cDNA是一条长链α-NT基因。 |
| 关键词: 眼镜王蛇 α-神经毒素 基因序列 |
| DOI： |
| 投稿时间：2001-02-01修订日期：2002-03-26 |
| 基金项目: |
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| Cloning and Sequence Analysis of the cDNA Encoding of α-Neurotoxin from Guangxi King Cobra (Ophiophagus hannah) |
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Li Qibin, Huang Guangwu, Zhao Xiaoqin, Y. Togesi, K. Kinjoh, M. Nakamura, T. Kosugi
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| (Snakebite Research Department, The first Affiliated Hospital, Guangxi Medical University, 6 Binhulu, Nanning, Guangxi, 530021, China) |
| Abstract: |
| 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. |
| Key words: Ophiophagus hannah α-neurotoxin cDNA sequence |
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