Prokaryotic Expression, Purification Of Anti-cyclinD1 Single Chain Fv And Analysis Of Activity

Cyclin D1 is the first cyclin synthesized when cells enter the cell cycle. It plays an important role in occurrance, development and prognosis of tumor. Cyclin D1 gene has been recognized as an oncogene and a potential target for cancer therapy.Single chain Fv is one of the gene-engineering antibodies. It has many of advantages, such as low molecular weight and high penetrability and possesses a great promise and potential in anti-cancer therapy.In present study, cyclin D1 was used as a target to get anti-cyclinD1 human-derived scFv. Then genes of scFv were expressed and the products were purified. The activity of scFv was determinated by ELISA, westernblot, Dot-blot, competitive inhibition, affinity constant determination and immunofluorescence dying.The results showed that seven phage antibodies were able to bind with cyclin D1 protein specifically. Three scFv genes showed soluble expression and the resulting scFv had binding activity after being transformation into E.coli HB2151. ScFv4 and scFv5 were purified by NI-NTA column successfully. ScFv4...