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Construction, Expression And Functional Characterization Of The RGD4C-β-Lactamase

Posted on:2013-01-11Degree:DoctorType:Dissertation
Country:ChinaCandidate:H WangFull Text:PDF
GTID:1114330374473759Subject:Radiation Medicine
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Chemotherapy is the most common treatment for patients with cancers. However, the dosage of most chemotherapeutic agents is limited by their severe side effects, which result in a very narrow therapeutic window. Antibody-directed enzyme prodrug therapy (ADEPT) has the potential to overcome this limitation by effectively bypassing systemic chemotherapy. ADEPT systems are based on fusion proteins or chemical conjugates that combine two domains:(a) an antibody fragment that binds selectively to tumor tissue, and (b) an enzyme domain that catalyzes the conversion of a nontoxic prodrug to an active drug at the tumor site. This site-specific activation of the prodrug results in a high local concentration of the chemotherapeutic agent in tumor tissues while minimizing exposure of healthy tissues. ADEPT systems have been described based on a variety of different enzymes. The targeted delivery of β-Lactamase to a vascular proliferation antigen (e.g., angiogenic vessel endothelium markers) is one relevant possibility.The RGD4C peptide has been proven useful for delivering various antitumor compounds, such as chemotherapeutic drugs and proteins to tumor vessels. This peptide binds to an av integrins, which expressed by angiogenic vessels and surface of tumor cells.By using gene recombination technology, a cloning vector was successfully constructed and the RGD4C-β-Lactamase (RGD4CβL) was subcloned into a bacteria expression vector. The expression product was obtained as well, with RGD4C in the N-terminal of β-Lactamase. We investigated its activity, immunogenicity, targeting; synthesized its prodrugcephalosporin-melphalan and investigated the effect on MCF-7of RGD4CβL-prodrug system.This study cloned a fused cDNA and successfully expressed active recombinant protein in E. coli purified with Ni-NTA resin. After cleavage and purification,β-Lactamase moiety showed the expected size of42kDa on SDS-PAGE, and was further confirmed by Western blotting. Using nitrocefin as substrate, the fusion protein showed good activity, and stability in human plasma; it also showed low immunogenicity in human PBMC and mice splenocytes proliferation assays.Based on flow cytometric analysis, the fusion protein was found to be specificity active for MCF-7cells. Whole body imaging and tissue distribution assay showed that the fusion protein has targeting effect to A549and U87MG cells.In vitro study showed that RGD4C β L has the catalytic effect on its prodrug cephalosporin-melphalan. the fusion protein and prodrug both showed low cytotoxicity on MCF-7, and when they are used together, the system show nice effect on MCF-7, the effect equal to the melphalan in the same dosage.All the studies will lay the foundation for further in-depth activity experiments.
Keywords/Search Tags:ADEPT, RGD4C, β-Lactamase, Recombinant protein
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