Expression and characterization of fusion protein tTF/SA as a universal effector for targeting blood coagulation / 生物工程学报

<p><b>UNLABELLED</b>To prepare a novel fusion protein (tTF/SA) as a universal effector for targeting therapy of blood coagulation and to analyze its biological activities. The fusion gene tTF/SA was constructed by PCR, then inserted into expression vector pET22 b (+), and expressed in E. coli BL21 (DE3). The fusion protein was purified using Nickel-affinity chromatography column. The activities of tTF moiety of the fusion protein were analyzed by clotting assay and FX activation assay, and the binding activities of Streptavidin(SA) to Biotin(B) were analyzed using ELISA.</p><p><b>RESULTS</b>The recombinant plasmid tTF/SA/pET22 b (+) with the correct sequence was obtained. The fusion gene tTF/SA was expressed with high yield in E. coli BL21 (DE3). The purified fusion protein retain the abilities of activating FX, inducing blood coagulation, and binding Biotin. The fusion gene tTF/SA was successfully expressed in E. coli BL21 (DE3). The recombinant tTF/SA proteins retain the activities of TF and SA. The multitarget therapy of selectively inducing thrombosis in tumor blood vessels can be achieved by the combination of tTF/SA, as a universal effector, and biotinlated carriers directing to tumor blood vessels.</p>

Gene construction, expression and activities analysis of human leptin / 生物工程学报

Six 89bp primers were designed on the base of the cDNA sequence encoding the human leptin reported on the NCBI. The synthetic gene with 464bp encoding rhLep was obtained by SOE ( splicing by overlap extension) PCR. The expression vector pET22b(+)/rhLep was constructed and transformed into E. coli BL21 (DE3). The rhLep protein was expressed as inclusion bodies with the yield of more than 50% of total bacterial proteins after IPTG induction. The rhLep protein, which has a molecular weight about 16kD, was purified by Ni2+ affinity chromatography column and identified by SDS-PAGE. The MTT Assay shows that rhLep promotes EC304 cells growth at the low concentration of 10ng/mL to 30 ng/mL, and rhLep appears cytotoxic to EC304 cells with the high dose of 50ng/mL to 225ng/mL. The viability of EC304 cells decreases to 1.2% with the concentration of 225ng/mL of rhLep. The massive apoptosis of rhLep on EC304 cells is observed by AO-staining under fluorescent microscope. All these results would lay the foundation for the further study of its biological functions in vitro and in vivo.