ABSTRACT
Chinese cobra (Naja naja atra) cardiotoxins are three-fingered family with 60～62 amino acids bind by four disulfide bonds. CardiotoxinⅢ (CTXⅢ) is one of the major toxic component which can cause hemolysis and cytotoxicity. However, there is no report on the fusion expression of CTXⅢ in soluble form so far. The cloning, expression and purification of recombinant CTX Ⅲ (rCTXⅢ) from Naja naja atra in E. coli and in yeast Pichia pastoris were reported here. CTXⅢ gene, fused with enterokinase in E.coli His-patch Thioredoxin expression system, were expressed in soluble form and released by osmotic-shock treatment. CTX Ⅲ gene was also cloned and expressed in the methylotropic yeast Pichia pastoris pPIC9K expression vector in the first time. The yield of the secretion level was 9.5 mg/L. Using straightforward one-step chromatography procedure, the rCTXⅢ, with three additional amino acids (GYT) at the N-terminal site, was purified to a purity of more than 90% and recovery yield of 65%. The purified rCTX Ⅲ was further characterized by cytotoxic assay with IC50 4.66μg/ml. An effective expression and purification system for recombinant CTXs in P. pastoris was developed, this system will permit us the ready isolation of active cardiotoxins. This protocol can also be easily used for the production of the toxin in a larger scale with low cost.
ABSTRACT
Objective To study the anti-lipid peroxidation effect of seal oil on rat liver steatosis induced by carbon tetrachloride and high fat diet and its mechanism of action. Methods: Both sc a low dose of carbon tetrachloride and high fat diet were given to male Wistar rats for 7 weeks . Then five groups(n=10 in each) received olive oil, simvastatin 4 mg ? kg-1 ? d-1, seal oil 0. 5, 1. 6, 4. 8 g ? kg-1 ? d-1 , administered orally for further 8 weeks,respectively . The untreated control group received only normal feed. The efficacy of seal oil on fatty liver and anti-lipid peroxidation was observed. Results: The contents of MDA and FFA decreased, CYP2E1 expressed weakly and SOD increased significantly in seal oil groups while hepatic TC,TG decreased and fatty liver was markedly improved when tested by pathologic diagnosis. Conclusion: Seal oil can induce SOD activities and eliminate oxygen free radical, thus show anti-lipid peroxidation effects on experimental fatty liver.