Research On The Separation And Purification And The Analgesic Effect Of Guangxi Cobra Neurotoxin

AIM: To separate, purify neurotoxin in Guangxi cobra venom and study the physical and chemical properties of neurotoxin as well as the analgesic effect of neurotoxin and NT -Tf conjugate .Method: To take snake venom through CM Sephadex C-25 and Sephadex G-50 gel filtration. The purified product was identified by polyacrylamid gel electrophoresis;the relative moleculer weight was determined by SDS-polyacrylamid gel electrophoresis. In order to separate and purify transferrin from SD rat blood serum, ammonium sulfate salting out and DEAE Sephadex A-50 ion exchang chromatography were adopted. The purified transferrin was identified by polyacrylamid gel electrophoresis. Neorotoxin was linked with transferrin by 2-iminothiolane and m-Maleimidobenzoyl-N-Hydoxy-succini-mide ester (MBS) respectively. NT-Tf conjugate was synthesized. The purified transferrin was identified by Sephacryl S-200 gel filtration chromatogram. The Tf of NT-Tf conjugate was measured by on-way immunodiffusion assay. Hot plate test and mouse writhing test were carried out to study the antinociceptive activity and mechanism of NT.Result: The sepatated NT was verified to be homogeneous .Its molecular weight was 7862Da.The pain threshold increase was 53.4% by i.v. NT, and 95.1% by i.v. NT-Tf conjugate. The writhing inhibition rat was 57.6%.The antinociceptive effect caused by intravenous injection of NT-Tf conjugate was partly blocked by systemic administration of atropine (p<0.01) ,however, the same effect caused by intravenous injection of NT-Tf conjugate was not blocked by methylatropine (p>0.05) .Conclusion: After two column chromatographies NT was separated and purified. NT was transported to pass through the blood-brain barrier by NT-Tf conjugate and could cause strongly antinociceptive effect in central nervous system.