中国生物工程杂志

The principal purpose of this study is to set up the isolation methods for diphtheria toxin expressed by genes, to purify the toxin with complete biological activities, and to make researches on its toxicities. In the experiments, the target gene was inserted into the cloning vectors--pET22b, then the vectors were introduced into host cells -BL21(DE3). The expression of the toxin was induced by addition of IPTG. The expression products were purified by His-6 label affinity chromatography. Then the diphtheria toxin expressed was transferred to PVC membrane using semidry blotter to determine the amino acid sequence of N-terminal. The "nicked" diphtheria toxin linked by disulfide bond was gained by protease hydrolysis. At the same time, acute toxicities of diphtheria toxin in guinea pig, cytotoxicity in three kinds of cells were tested. The results indicated that the expression system established in the study expressed DT effectively. Amino acid sequencing of expressed DT gave the N- terminal sequence- MGADDVV…. This was identical with the experiment project. The LD50 of DT in guinea pig (ig) was 0.64μg/kg; and the IC50 values of DT in CHO-K1, BS-C1 and Hela were 1.709μg/ml, 1.424ng/ml and 28.947ng/ml respectively.