Guangxi Cobra Venom Ngf And Natrin, Molecular Biology And Nature Study

Objective:Gene Cloning and Expression of NGFfrom Guangxi Cobra Venom(Naja naja atra).To study apoptosis induced by NGF on Noovel Human palatal salivary gland cells of adenoid cystic Carcinoma(NACC).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 ion exchange 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 pMD 18-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.The expressed product was purified by immobilized metal(Ni²⁺)chelation affinity chromatography.The expressed product was testified by Western blot.The activity of the expressed product was measured by observing the cell sharp and counting the cell number after adding this protein into PC 12 cell.Cell apoptosis were observed by flow cytometry and cell morphology.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.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.Western blot analysis indicated that the expressed protein cross-reacted with the antibody against the native enzyme.In addiction,the survival activity of expressed protein was proved by examing the survival rate of PC12 cells in serum-free medium.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.NGF from Guangxi Cobra Venom can induce apoptosis of NACC cell. Objective:Gene Cloning and Expression of Natrin from Guangxi Cobra Venom(Naja naja atra);To study the effect of Natrin on lipid peroxide of the rat liver induced by Fe²⁺;To study apoptosis induced by Natrin on MGC-803 cells.Method:The Natrin from Guangxi cobra venom(Naja naja atra)was sequentially purified by Sephadex G-50 gel filtration,CM-Sepharose CL-6B ion exchange 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 N-terminal and C-terminal amino acid sequences were synthesized,the PCR product was cloned into the pGEM-T vector.The complete sequence of Natrin was determined by sequencing and its amino acid sequence was deduced.The gene was cloned into the expression plasmid pMAL-c2x,after transforming into E.coli BL21(DE3),Natrin was induced to express by IPTG.The expressed product was purified by Amylose chelation affinity chromatography.The expressed product was testified by Western blot.Using MDA colorimetry,the content of MDA,the production of lipid peroxide of the rat liver's mitochondria was determined.Cell apoptosis were observed by flow cytometry and cell morphology.Result:The amino acide sequence which presents 92%,85%,86%,83%homology to that of Ophanin,Philodryas olfersi,Ablomin i and Trimeresurus respectively,encoded a mature Natrin molecule with 221 amino acides.Western blot analysis indicated that the expressed protein cross-reacted with the antibody against the native enzyme.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. Natrin had inhibited lipid peroxidation at higher concentration.Natrin from Guangxi Cobra Venom can induce apoptosis of MGC-803 cell.