Preparation And Experimental Study On Anti-tumor Effect Of DT₃₈₉-SS In Vitro

Cancers have been threatening the health of human beings. The chemicals used in clinical now lack selectivity. Toxical effects are common seen in clinical. Tumor-targeting therapy will be satisfactory for enhancing therapeutic effects of anti-tumor medicines.Somatostatin is a single chain peptide, which has two forms referred to as ss-14 and ss-28, reflecting their amino acid chain length. Somatostatin was found to be sreted by a broad range of tissues, including pancreas, intestinal tract and regions of the central nervous system outside the hypothalamus. The over-expression SSR which can be binded specially with SS was found on many cancer cells surface including neuroendocrine tumour, nervous system tumour and colon carcinomas. DipHtheria toxin (DT) is a single chain bacterial toxin, which is composed of three structural domains. Domain C is responsible for cell binding (amino acids 386~535), which can be replace the gene of antibody and cell factor. Domain T (amino acids 205~378) plays an essential role in the translocation of DT when it across the cell membrane and into the cytosol. Domain N is composed of domain (amino acids 1~193) and is responsible for catalytic domain with ADP-ribosy transferase activity can inactivate elongation factor 2 , and cause cell death.The gene fragment was amplified from PCDNA3-DTA by overlap extension PCR . The PCR products were digested by EcoRⅠand HindIII,and then inserted into plasmid vector pET28a(+). The positive recombinant plasmids were transformed into host strain E.coli BL21(DE3) and induced to express recombinant protein of DT389-SS by IPTG. The specific protein expressed (about 45.0kDa) was detected by SDS-PAGE. The protein was expressed at high level, amounting to 31% of the total bacterial protein as confirmed by the soft of computer scanning. Western blot analysis showed that the fusion protein may react specifically with anti-6×histag antibody.High cell density fermentation of the recombinant E.coli strain was studied, for optimizing the cell density and protein production. Several factors were investigated, such as the selection of host bacterium, the selection of medium, the selection of induced dose and optimized condition. In our study, the recombinant plasmid of DT389-SS can be express in four E.coli strain. After trial and error, we choose BL21(DE3)LysS strain. The optimized condition: the medium is LB, the pH is 7.0, the induced temperature is 23℃and time is 16 hours, the dose IPTG is 0.1mmol/L. Under induction of IPTG the recombinant protein was expressed as a soluble protein and was up to 12% of the total protein in E.coli BL21(DE3) LysS.Using glycerol as carbon sources, IPTG as inducing agent and specific fed-batch mode as feeding method while keeping stiring speed and DO at 20%, E.coli cell density could reach 30g/L and the expression remains at high level. the recombinant protein was expressed as a soluble protein and was up to 20% of the total protein in E. coli BL21(DE3) LysS.Cells was collected by centrifugation and lysed by sonication. Protein was purified by Ni+ chromatography. With the above purification, the purity of DT389-SS was about 95%. The cytotoxity of DT389-SS on SWWC and SPC-A1 cell growth was analyzed with MTT. The ID50 of DT389-SS on two cell were 16μg/mL and 15μg/mL.