蛇神经毒素的表达和鉴定

抽提中华眼镜蛇毒腺总RNA，通过反转录PCR扩增Cobrotoxin cDNA,克隆并测序。该cDNA编码83个氨基酸，包括21个氨基酸的信号肽和62个氨基酸的成熟蛋白。该成熟蛋白的氨基酸序列和通过蛋白测序从台湾眼镜蛇鉴定的Cobrotoxin完全一致。PCR扩增编码Cobrotoxin的DNA，并亚克隆到表达载体pMAL-P₂。此外，通过合成寡核苷酸片段，拼接成完整的CM11基因，并将其克隆至pMAL-P₂。经IPTG诱导，两种神经毒素基因在大肠杆菌中都得到高效的可溶性融合表达。表达产物通过SDS-PAGE和蛋白印迹杂交加以鉴定。表达的融合蛋白经过Sepharose 6Bamylose亲和色谱和DEAESepharose FF离子交换色谱得到有效纯化。经Xa因子酶切后得到的两种重组神经毒素都具有小白鼠体内毒性。

Expression and Characterization of Two Kinds of Recombinant Snake Neurotoxins

The cDNA encoding the precursor of cobrotoxin was cloned from the venom gland of the Chinese continental cobra ( Naja naja atra ) by RT PCR.Its deduced amino acid sequence analysis showed that the mature protein was identical to that identified from the Taiwan cobra ( Naja naja atra ) by protein sequencing technique.The cDNA encoding the mature protein was then subcloned into the expression vector pMAL P 2.The gene of CM11,which was formed by ligation of the fragments of the synthetic oligonucleotides,was also cloned into the expression vector pMAL P 2.After induction of IPTG,both of the neurotoxins were overexpressed as soluble fusion proteins which were confirmed by SDS PAGE and western blotting.The expressed fusion proteins was purified by sepharose 6B amylose affinity chromatography and DEAE sepharose FF chromatography.Both of the recombinant proteins achieved after digestion by factor Xa showed the in vivo toxicity.