Gene Cloning And Expression Of NGF From Guangxi Cobra Venom

Objective: Gene Cloning and Expression of NGF from Guangxi Cobra Venom (Naja naja atra).Method: The nerve growth factor (NGF) from Guangxi cobra venom (Naja naja atra ) was sequentially purified by Sephadex G-50 gel filtration CM-Sepharose CL-6B ionexchange chromatography and Mono S column chromatography on FPLC system. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted from the snake venom gland,two oligonucleotides primer deduced from the most stringent conserved regions were synthesized, the PCR product was cloned into the pMD18-T vector.The complete sequence of NGF was determined by sequencing and its amino acid sequence was deduced.Then degenerate primer was desidned and synthesized according to the N-terminal amino acid sequence as the mature NGF DNA. The gene was cloned into the expression plasmid pET-40b(+), which carries a His Tag sequence.After transforming into E.coli BL21(DE3),NGF was induced to express by IPTG. Result: The amino acide sequence which presents 99%,91%,90%,66% and 68% homology to that of Naja kaouthia (monocled cobra) Bungarus multicinctus Crotalus durissus terrificus (tropical rattlesnake) mouse and human NGF respectively,encoded a prepro-NGF molecule with 242 amino acides and a mature NGF molecule with 116 amino acides . Western blot analysis indicated that the expressed protein cross-reacted with the antibody against the native enzyme. According to the deduced amino acide sequence,its molecular weight and PI are determined to be 12346.93 Daltons and 7.643 respectively as calculated by DNAclub and DNAstar softwares.These would provide a good basis for further discussing the relation of structure and function ,and the studying of evolution of NGF from snake venom.