蛋白质组学研究的主要策略及其在肿瘤研究中的应用

恶性肿瘤无限增殖的主要原因是细胞凋亡调控障碍。Ｓｕｒｖｉｖｉｎ是新近发现的凋亡抑制蛋白家族 (ｉｎ ｈｉｂｉｔｏｒｏｆａｐｏｐｔｏｓｉｓｐｒｏｔｅｉｎ ,ＩＡＰ)成员1] ,特异性表达人类多种常见肿瘤中 ,而不存在于正常成人组织 ,能抑制ｃａｓｐａｓｅ活性而发挥抗凋亡作用2 ] 。有研究表明3 4 ] ,应用反义策略阻断ｓｕｒｖｉｖｉｎ表达可提高肿瘤细胞对化疗药物的敏感性 ,诱导凋亡的发生 ,已成为肿瘤治疗的新亮点。本文构建了ｓｕｒｖｉｖｉｎ反义ＲＮＡ真核表达载体 ,为降低肿瘤细胞ｓｕｒｖｉｖｉｎ的表达 ,进一步研究肿瘤细胞…

Construction of the Tetramerizing Single Chain Fv Antibody Gene Specific for Human Prostate Specific Antigen and Its Expression in HeLa Cells

Objective: To construct anti human prostate specific antigen (PSA) single chain Fv antibody (scFv)/human p53 tetramerization domain fusion gene and express fusion protein in HeLa cells. Methods: The human IgG3 upper hinge/human p53 tetramerization domain fusion gene was obtained by recursive polymerase chain reaction (PCR), and was inserted into pUC19 to construct cloning plasmid pUC19/IgG3/p53. The anti PSA scFv was then cloned into pUC19/IgG3/p53 to construct anti PSA scFv /human p53 tetramerization domain fusion gene which was then subcloned into the pSecTag2 B expression plasmid. Then the pSecTag2 B plasmids concluding the fusion gene were transfected HeLa cells. The expression products were analyzed by both SDS PAGE and Western blot, then were purified with Ni 2+ NTA superflow affinity chromatography. The binding affinity for PC 3 cells was measured by flow cytometry. Results: The anti PSA scFv/human p53 tetramerization domain fusion gene consisted of 891bp encoding 297 amino acid residues, and was the same as that reported before. The expression products of the tetrameric anti PSA scFv, which relative molecular mass (Mr) was about 35 000, were confirmed by SDS PAGE and Western blot. After purified with Ni 2+ NTA superflow affinity chromatography, the tetrameric anti PSA scFv showed significantly stronger binding to PC 3 cells than scFv.Conclusion: The tetrameric anti PSA scFv which could bind to PC 3 cells has been successfully gained for the potential use in clinical studies.