血管生成抑制因子融合蛋白抗肿瘤作用的基因治疗研究

Until now, many antiangiogenesis agents have been reported, but only about 30.of them are in the clinical research, and none of them has got the pharmaceutical.certificate. Cartilage is the most important avascular tissue in the body. Cartilage contains angiogenesis inhibitory factors has been reported by several lab..Shark cartilage angiogenesis inhibitory factor-I (SCAIF-I) was purified to.homogeneity. The inhibitor, extracted from shark cartilage using 4 M Gu·HCI, was.fractionally precipitated with 35%~65% acetone. Further purification was achieved by Resource Q ion exchange chromatography, Sephacryl S-300 gel filtration, and.reverse-phase HPLC. The inhibitor was judged homogeneous by the appearance of a.single band on a silver-stained 15% sodium dodecyl sulfate-polyacrylamide gel..SCAIF-I had an Mr of 18 KD. NH2-terminal sequence data were obtained for the first.27 residues (Tyr-Thr-Tyr-Gln-Lys-Lys-Glu-Leu-Ala-Arg-Val-Leu-Gln-Ser-Lys-Gly-.Leu-Asp-Gly-Tyr-Arg-Gly-Tyr-Ser-Leu-Ala-Asn). SCAIF-I specifically inhibits.movement and proliferation of endothelial cells; and angiogenesis in the.chorioallantoic membrane of chick embryos. Systemic administration of SCAIF-I at.the dose of 5 mg/kg.d suppressed the growth of primary Lewis lung carcinoma.implanted in C57BL/6 mice by 87.93%..A novel inhibitor of angiogenesis named as shark cartilage angiogenesis.inhibitory factor-II (SCAIF-II) from shark cartilage was isolated by the method.similar to that of SCAIF-I. SDS-PAGE analysis followed by silver staining revealed a single band having a molecular weight of 80 KD. To determine whether this protein was capable of inhibiting angiogensis, it was assayed in capillary endothelial cell (EC) proliferation and migration detection. The results showed SCAIF-II significantly suppressed capillary EC proliferation and migration in a dose-dependent. To determine whether SCAIF-II was an inhibitor in vivo, it was detected in the chick chorioallantoic membrane (CAM) assay, and the result showed that SCAIF-II inhibited angiogenesis in chick CAM potentially. In animal studies, the growth of.tumor was potently suppressed by SCAIF-II treatment. Lewis lung carcinoma was.inhibited by 93.83 % at a dose of 10 mg.kg-1.d-1. These findings suggest that shark cartilage produce another novel protein with anti-angiogenic and anti-tumor activity, which should be a useful source of the inhibitor..The antibody of SCAIF-I was prepared by ordinary method from New Zealand.rabbits. With the antibody, the quantity of SCAIF-I in different tissues of shark and bovine was detected by the method of western blot. The results showed that SCAIF-I was only existed in shark cartilage. According to the N-terminal amino acid sequence of SCAIF-I, the specific primers and degenerate primers are designed using the GCG package. Using PCR, the genome fragments was obtained. The fragments were cloned into pGEM-T easy.vector using pGEM-T easy vector system I according to the manual. The recombinant.plasmid was transformed E.coli DH5α, and the recombinants were identified and.7 sequencing. The result sequences were analyzed by GCG package, and selected the.sequence which was most fitted the N-terminal sequence of SCAIF-I..The fragments of PF4(58-70) and TSP1(429-459) were more powerful to inhibit angiogenesis than their intant proteins, respectively. TSF was designed containing PF4(58-70) and TSP1(429-459) that were ligation with Gly-Pro-Gly bridge. TSF gene was divided into 8 fragments to be synthesized, and the synthesized fragments were ligated and cloned into pUC118 plasmid to sequence. Then, TSF gene from the correct sequence of pUC118/TSF was cloned into a pGEX-2T expression vector to generated recombined plasmid—pGEX2T/TSF, and transformed into attenuated E.coli JM109 to construct recombined JM109(pGEX2T/TSF) strain. TSF was expressed highly in inclusion, and the quantity of TSF was 28% in the total of E.coli proteins. The purification procedure was established, and the purified TSF and its fuse protein GST-TSF were obtai

已经纯化鲨鱼软骨血管生成抑制因子（CAIF）并获得其cDNA，表达得到高效低毒的CAIF。本项目对其进行基因治疗研究，合成PF4C-端片断及TSP1的N-端片断基因，与CAIF基因组合成诤匣颍≡裣傧喙夭《咀髟靥澹迦隒MV启动子，构建重组病毒，进行体内外的试验，筛选出高效重组子，进行抗血管及抗肿瘤试验。为肿瘤的抗血管疗法提供一种高效价廉的新途径。