Purification Of Nerve Growth Factor From Guangxi Cobra Venom By Affinity Chromatography And Its Study On Antitumor Function

Objective : In order to shorten the purification cycle of NGF,and increase the yield of NGF,affinity chromatography was used to purify nerve growth factor(NGF) from Guangxi Cobra Venom , which was subsequently investigated about its growth inhibitory function on human tumour cells.Methods: (1)The crude snake venom was separated and purified by gel filtration on a Sephadex G-75 colum and CM-Sepharose CL-6B cation-exchange collumn.The activity of the eluting peak was assayed by diphasic immunodiffusion and rat pheochromocytoma cell ( pc12 cells) ,and then the purity and the relative molecular mass of the obtained NGF eluting peak were identified through sodium-dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE). (2) Antiserum gainst NGF was separated from rabbit immuned by NGF which showed one band by SDS-PAGE, then the IgG in the antiserum was purified by ammonium sulfate precipitation method and HiTrap rProtein A FF collumn.Purified IgG was coupled to CNBr -activated Sepharose 4 Fast Flow .Then NGF was purified from Guangxi Cobra Venom by affinity chromatography.(3) The cytotoxicity and the growth inhibition ratio of four kinds of human tumor cell lines , MGC-803,SGC-7901,BEL-7404 and TCA-8113 were treated with NGF at different final concentrations of 7.5,15,30,60 and 120mg/ L for 24h were assayed by MTT colorimetric method. (4) The tinction and morphology of MGC-803 cells treated with NGF at different final concentrations of 25,50 and 75mg/ L for 24h were observed by acridine orange/ethidium bromide (AO/EB) double staining.(5) The rate of apoptosis was detected by flow cytometer ( FCM) which doublely tagged by Annexin V-FITC / PI.Results : (1) NGF was purified successfully from Guangxi Cobra Venom by affinity chromatography.The product was proved to be homogeneous and SDS-PAGE showed a single strap. Its relative molecular mass is approximately 23 000, and it can promote the differentiation of PC12 cells favourably .(2) NGF had obvious cytotoxicity on these cell lines ,which shows to be dose-dependent, especially for MGC-803 and BEL-7404 ,their IC₅₀ value at the 24^th hours are 49.9mg/L and 55.46mg/L ; Main features of apoptosis under fluorescent microscope include reduction in volume,nuclear chromatin condensation, intensification under jacinth fluorescence; The early apoptosis rate of pristine cells which detected by FCM increases with NGF dose increasing.Furthermore,the early apoptosis rate of each concentration significantly differ from the control(P<0.01).Conclusion : (1) It shown that NGF separation by affinity chromatography is simpler than that by traditional purification.It only need one-step,which consumedly decurtate the purification cycle of NGF, and increase the yield of NGF .This method was technical support for purifying NGF in great scale (2) NGF possesses distinct cytotoxicity and growth inhibitory effect on the four kind of human tumor cells especially on MGC-803 and BEL-7404, inducing apoptosis may play an important role in its mechanism.