Expression, Purification And Functional Study Of Recombinant Human Plasminogen Kringle 5 With Yeast Pichia Pastoris And Antiangiogenic Gene Therapy Of Tumor

Tumor angiogenesis is a hallmark of cancer and palys a pivotal role in tumor growth and metastasis. Various studies have demonstrated that drug based antiangiogenic therapy and antiangiogenic gene therapy are effective in the treatment of cancer, which will become a prospective adjuvant anticancer therapy.Recently, a great number of endogenous angiogenic inhibitors have been discovered, such as Angiostatin and Endostatin etc, which inhibited endothelial cell proliferation, migration and tube information, experimental and clinical studies have proved that these substances inhibit tumor growth, metastasis and angiogenesis. Angiostatin is the N terminal fragment of plasminogen, which consists of four kringle domains of plasminogen molecule. It has been shown that kringle domain is essential for the antiangiogenic activity of Angioststin. Analogues of angiostatin such as kringle 1-3 (K1-3) and kringle 5 (K5) possess similar antiangiogenic activity and K5 is a potent inhibitor of endothelial cell proliferation, migration and angiogenesis.To further investigate the antiangiogenic activity of human plasminogen kringle 5 (K5), first, we constructed and expressed K5 cDNA clone with yeast Pichia pastoris, purified the recombinant protein (rh-K5) and tested its anti-proliferation effect on bovine capillary endothelial (BCE) cell; second, we constructed an eukaryotic expression plasmid pcDNA3K5 and recombinant replication deficient adenovirus K5 (Ad-K5); assayed the effect of K5 cDNA gene transfer on the proliferation of BCE, human umbilical endothelial cell ECV304 and human breast carcinoma cell MDA-MB-231, the tube formation of BCE and ECV304 cells on ECMatrix gel; as well as the gene therapy of K5 which was evaluated on a BALB/c nu/nu MDA-MB-231 breast carcinoma model.Section 1 Expression, purification and biological activity of recombinant K5 The human plasminogen kringle 5 cDNA was amplified via PCR and recombined with Pichia pastoris expression vector pPIC9K, the recombinant clone pPIC9KK5 was picked up and transformed yeast Pichia pastoris, the transformants were screened and cultured by methanol induction, expression product rh-K5 was purified and tested its antiproliferation effect on BCE cells.We constructed a recombinant vector pPIC9KK5 with only one protesase cleavagesite (Kex2) preserved in the α-factor signal peptide in order to get uniform N terminal of rh-K5. The pPIC9KK5 was then transformed yeast Pichia pastoris, the transformants were screened and their phenotypes were identified by PCR. The positive clones were cultured by methanol induction. The secreted expression product rh-K5 was verified by SDS-PAGE and Western Blot, purified by Sephadex G-50 gel filtration. It was shown that the purified rh-K5 inhibited r-bFGF (recombinant basic fibroblast growth factor, r-bFGF) induced proliferation of BCE cells by enhancing apoptosis, but had little effect on cell cycle.Section 2 In vitro study of K5 cDNA gene transferThe human plasminogen signal sequence was fused in-frame with K5 cDNA via PCR, and recombined with eukaryotic expression vector pcDNA3, the recombinant plasmid pcDNA3K5 was transfected human breast carcinoma cell MDA-MB-231. Positive clones were screened by G418 and identified by PCR, RT-PCR and Westeren Blot. The conditioned media from MDA-MB-231 cells transfected with pcDNA3K5 were applied to BCE and ECV304 cells to test its antiproliferation effect. K5 cDNA cut from pcDNA3K5 was recombined with pShuttle vector, and then integrated into replication defective Adenovirus DNA, the recombinant replication defective Adenovirus K5 plasmid DNA (pAd-K5) was transfected HEK293 cells, packaged and amplified the virus within the cells. The recombinant replication defective Adenovirus K5 (Ad-K5) was prepared and infected BCE, ECV304 cells and MDA-MB-231 cells to assay its antiproliferaction effect, as well as the tube formation of BCE and ECV304 cells on ECMatrix gel.The recombinant plasmid pcDNA3K5 was verified by endonuclease analysis and DNA sequencing, which sh...