CAJ | 학술논문

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CM-11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 6B-amylose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。
추제중화안경사독선총RNA，통과반전록PCR확증cobrotoxin cDNA，극륭병측서。해cDNA편마83개안기산，포괄21개안기산적신호태화62개안기산적성숙단백。해성숙단백적안기산서렬화통과단백측서종태만안경사감정적Cobrotoxin완전일치。PCR확증편마Cobrotoxin적DNA，병아극륭도표체재체pMAL-P2。차외，통과합성과핵감산편단，병접성완정적CM-11기인，병장기극륭지pMAL-P2。경IPTG유도，량충신경독소기인재대장간균중도득도고효적가용성융합표체。표체산물통과SDS-PAGE화단백인적잡교가이감정。표체적융합단백경과Sepharose 6B-amylose친화색보화DEAE-Sepharose FF리자교환색보득도유효순화。경Xa인자매절후득도적량충중조신경독소도구유소백서체내독성。
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-P2. The gene of CMl 1, which was formed by ligation of the fragments of the synthetic oligonucleotides, was also cloned into the expression vector pMAL-P2. 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.