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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。
In order to obtain the gene sequence of α-neurotoxin (α-NT) of Ophiophagus hannah (Oh) snake venom, we designed a pair of primers based on the higher homology of α-NT gene from different serpent species In order to overcome the ambiguous amplification caused by the primers, a pair of upstream and downstream specific primers were designed in the protein coding region. The m RNAs were extracted from 3 live cobra venom glands by Nacleospin RNA Kit and synthesized by 3 ’ c DNA as a template for PCR amplification, determination of the nucleotide sequence of the product, to obtain a full-length 4 74 bp of King Cobra c DNA gene nucleotide sequence. The signal sequence of this nucleotide sequence was 100% homologous with the Pseudonnaja textilis (Pt) and the sea snake L aticauda semif asciata (L s), and was highly homologous to the cobra snake N aja sputatrix (Ns), Bungarus multicinctus ) (Bm) was 96.8% homologous; the protein coding part was 83.3% homologous with Ns, 79.2% with Pt, 76.4% with L s and 74.1% with Bm. Ninety-three percent of the 72 amino acids immediately after the signal peptide were homologous to the long-chain alpha-NT Toxin a of the pokeweed, about 73.6% with Toxin b, 67.7% with Oh-4,66. 7% and Oh-5,5 6 .9% are homologous to Oh-6 A and 6 B and homologous to 54.2% of α-bungarotoxin. Description of the newly discovered king cobra c DNA is a long-chain α-NT gene.