Cloning and expression of the tumstatin active peptides-T(7) and its derivant-T(7)-NGR.

To enhance the role targeting, design to link NGR sequence with tumstatin active peptides-T(7)'s C-terminal, the derivant called T(7)-NGR. The cloning vector pMD-T(7) and pMD-T(7) N were constructed by PCR and gene synthesis methods, respectively, identified by digestion and DNA sequencing. After the digested plasmids were isolated by the low melting point agarose electrophoresis, the target-fragment was cut off and mixed with the recovery of the digested vector pET28a. Expression vector pET-T(7) and pET-T(7) N were constructed in low melting point agarose, identified by digestion and DNA sequencing, transformed into competent Escherichia coli BL21 (DE3), induced by IPTG. Identification result shows that pET-T(7) and pET-T(7) N were correct. Tricine-SDS-PAGE results showed that IPTG concentration of 1 mM, after the induction of 25 degrees C, 8 h, T(7) peptides and T(7)-NGR peptides have achieved the optimum conditions of expression. In conclusion, the expression vectors of the two peptides has been successfully constructed, and got product, no coverage at home and abroad, laid the foundation for further activity experiments.