| Anti-bacterial peptide (abbrevitated form-ABP) is a kind of induced small size molecular peptide which has important biological activity in organism. Its molecular weight is from 2000 to 7000, consists of 20 -60 amino acids. Cecropin B is isolated from the giant silk worm for the first time by Boman. It is a dissoluble peptide which is thermally stable and broad-spectrum antibiotic and can be induced in organism. Cecropin B is not only useful in the aspect of antibiosis but also good at killing some kinds of tumor cells such as leukemiacell, cervical carcinoma, rectal carcinoma and so on. But it has no effect on normal ^mammalian cells and insect cells. Angiostatin is the degradated fragment of plasminogen and mainly consists of four fabric areas of plasminogen. Cao finds the fifth fabric area of plasminogen (plasminogen kringle5,hPgnK5) which has stronger antineoplastic activity and the ability of inhibiting vascular endotheli ocytosis. The generating, developing and shifting of the normal tumor heavily depend on the generating of new vasculare. Because of the inhibit function of Kringle5 to the generating of new vasculare, the growth of tumor vasculare is strongly inhibited. It strongly inhibits the growth of tumor cells through inhibiting the growth of tumor vasculare, makes tumor tissue atrophy and vanish.After Kringle 5 (K5) gene was isolated from plasmid ThioA/L15-7, the full-length antibacterial peptide cecropin B gene (CB) was artificially synthesized through using gene splicing by overlap extension (gene SOEing) method and was genetically fused to the 5' end of K5 gene, and then the fusion gene CB-K5 was subcloned into expression plasmid pET32a and the termination codon was designed at the 3' end of K5 gene, the recombinant expression plasmid pET32a/CB-K5 can be obtained. By using PCR and sequence analyses, it can be proved that all the tested clones contained the recombinant plasmid pET32a/CB-K5.The novel fusion expression plasmid pET32a/CB-K5 was successfully constructed.The fusion protein is efficiently expressedafter IPTG induction as a 37kD band on SDS-PAGE.The next step work that needs to be done is more deeply researching the antineoplastic activity of the fusion protein. The final target is to find out one more powerful medicinal recombinant protein, which will be the foundation of the further study. |
