PCR法分子克隆及序列分析广西产眼镜王蛇蛇毒α-神经毒素基因

摘要

目的为了获得眼镜王蛇(Ophiophagus hannah,Oh)蛇毒α-神经毒素(α-Neurotoxin, α-NT)的基因序列.方法我们通过比较了基因库中已知眼镜蛇科不同种类毒蛇来源的α-NT基因,发现它们有较高的同源性,特别是5'和3'非翻译区及引导肽部分高度保守,据此设计了包括翻译起始点的上游引物,以及为了得到3'端较完整非编码部分而设计了基本上属于d(T)的反意链下游RACE-PCR引物.为了克服引物所带来的模糊扩增,还在蛋白编码部分再设计一对上下游引物,由此组成P1、 P2、 P3和 P4四对引物.采用Nacleospin RNA Kit法分别从3条活Oh蛇毒腺中提取mRNA, 以3'端引物合成cDNA的第一链,并以此作为模板,分别用四对引物进行PCR扩增反应,得到了目的基因不同长度的PCR产物,产物经精制后进行测序,对比分析其结果.结果获得了全长474 bp的Oh.cDNA的基因核苷酸序列,包括5'端60 bp,信号肽伴启动子ATG 63 bp,蛋白质密码部分216 bp和3'端186 bp并含有TGA终止码.经基因库信息计算机分析其信号肽与眼镜蛇树属(Pseudonnaja textilis,Pt.)海蛇(Laticauda semifasciata,Ls)100%同源,96.8%与眼镜蛇南洋亚种(Naja sputatrix,Ns)和银环蛇(Bungarus multicinctus,Bm)同源.蛋白密码部分 83.3%与 Ns,79.2%与 Pt, 76.4%与 Ls和 74.1%与 Bm同源.氨基酸顺序分析信号肽后紧接着的72个氨基酸90.3%与已发现的眼镜王蛇毒长链α-NT Toxin a同源,大约 73.6% Toxin b 、69.7% Oh-4 、66.7% Oh-5 、56.9% Oh-6A 和6B同源,并与α-银环蛇毒素54.2%同源.结论新发现的Oh.cDNA属于长链α-NT的基因.%Objective To obtain the cDNA and sequence analysis of α-neurotoxin (α-NT) from Guangxi king cobra (Ophiophagus hannah，Oh)．Methods　Comparative analysis of the determined cDNA sequences of α-NT from elapidae snake venoms showed that the nucleotide sequences of 5'，3'-non-coding regions and the signal peptide coding region were highly conserved．Thus the sense primer was designed from 5' conserved regions， which contain the start codon ATG． The antisense primer d (T) was used for amplification of cDNA 3end (RACE-PCR)．Two primers were designed from the 5'， 3'- coding regions for overcome the mistake amplification by the primer．4 couple primers of P1， P2， P3 and P4 were designed from those 4 primers．Venom gland RNA was extracted from three snakes of Oh with isolation kit． The extracted RNA was then reverse transcribed into cDNA with oligo (dT) antisense primer． After RT-PCR with the designed primers the 4 fragments of PCR product isolation with electrophoresis were achieved． The nucleotides sequence analysis of fragment was on the ABI PRISM 310 automatic DNA sequencer．Results　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． Comparison of the cDNA of long chainα-NT between Oh snake and other snakes found the nucleotide sequence much higher corresponding to the signal peptide than the mature protein coding regions which is about 100% identical to Pseudonnaja textiles(Pt) and Laticauda semifasciata(Ls)，96.8% to Naja sputatrix(Ns) and Bungarus multicinctus(Bm)．Encoding 93 amino acid residues containing a signal peptide (21 amino acid residues) and Oh-toxin (72 amino acid residues) with 10 cysteine residues could be determined．The conservation of mature protein region about 83.3% to Ns，79.2% to Pt，76.4% to Ls and 74.1% to Bm snakes．It was found that the among conservation of Oh-toxin from cDNA，90.3% was identical to Oh Toxin a， about 73.6% to Oh Toxin b， 69.4% to Oh-4，66.7% to Oh-5， 56.9% to Oh-6A and Oh-6B；and 65.3% identical to LNTX， 61.1% to toxin a and 54.2% to α-bungarotoxin．Conclusion The result indicates that newly found Oh cDNA is a long chain α-NT gene．