TAT-PE Fusion Protein For Targeted Cancer Therapy

The malignant tumor seriously threatens health of mankind. The commonly used cancer therapy methods include surgery, radiotherapy and chemotherapy. However, the side effect of radiotherapy and chemotherapy is enormous, because the tumor cells and normal cells were simultaneously killed during treatment. Biotherapy is a new method for targeted cancer therapy, and it is a promising research field.One strategy for targeted cancer biotherapy is to utilize recombinant immunotoxins. Recombinant immunotoxins are composed of a very potent protein toxin fused to a targeting moiety such as recombinant antibody fragment or growth factor. The truncated version of Pseudomonas aeruginosa exotoxin A is widely used to construct immunotoxins.The potent clinical efficacy of immunotoxins have been demonstrated in patients with malignant diseases, and the recombinant immunotoxin ONTAK(DAB389IL2) has been approved by the FDA for CTCL therapy. But, the in vivo antitumour activity of recombinant immunotoxins is influenced by many factors, e.g. large size of the proteins and their poor ability to penetrate into tissues and tumours. Efforts are now being made to improve the current molecules and to develop new agents with better clinical efficacy. One approach to circumvent these problems is the use of HIV TAT-mediated bacterial toxin. The HIV Tat protein transduction domain (PTD) is a small basic cell-penetrating peptide(CPPs) that has been successfully shown to deliver large variety of cargoes, including peptides, proteins, and nucleic acids into cells. To reduce its toxicity to normal tissues, the TAT-mediated toxin can be injected directly into tumor tissues.In this dissertation, the following experiments are conducted:1. Cloning and expression of P. aeruginosa exotoxin A and its cytotoxicity / antitumour activities determination.â‘ After PCR conditions were optimized, the exotoxin A structural gene was successfully amplified from chromosomal DNA of Pseudomonas...