中华眼镜蛇神经毒素cDNA的克隆及表达

通过ＲＴ－ＰＣＲ的方法，从广西产中华眼镜蛇总ＲＮＡ中克隆到３个新的神经毒素ｃＤＮＡ序列，命名为ＮＬ１、ＮＬ２和ＮＬ３。它们编码的蛋白质序列与台湾眼镜蛇神经毒素ｃｏｂｒｏｔｏｘｉｎ的同源性分别为７７％、７２％和９８％，均具有维持ｃｏｂｒｏｔｏｘｉｎ结构与功能必需的残基Ｔｙｒ＾２５、Ｌｙｓ＾２７、Ｔｒｐ＾２９、Ａｒｇ＾３３和Ｌｙｓ＾４７。ＮＬ３克隆于ｐＥＴ２８ｂ＋表达载体内，转化至ＢＬ２１（ＤＥ３）中

Cloning and Functional Expression of Neurotoxin cDNA from Naja naja atra

Three new cDNAs named NL1, NL2 and NL3 were cloned from the total RNA of Naja naja atra by RT PCR. The protein sequences encoded by them showed 77%, 72% and 98% structure identity to cobrotoxin which is a postsynaptic neurotoxin from Taiwan cobra( Naja naja atra ), respectively. The five conservative residues, Tyr 25 , Lys 27 , Trp 29 , Arg 33 and Lys 47 , essential for the function of cobrotoxin were also found in the three cDNAs. The NL3, the most homologous to cobrotoxin, was expressed in E.coli BL21(DE3) by cloning into pET28b . The expressed product was insoluble inclusion bodies and could be purified up to 90% purity in the range of 6 mg per liter culture cells by a single affinity step. The purified protein was refolded in vitro and the toxicity was assessed to be less than the native cobrotoxin in mice.