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Date of Award

2007

Document Type

Dissertation - Pacific Access Restricted

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

Xiaoling Li

First Committee Member

Bhaskara R. Jasti

Second Committee Member

William K. Chan

Third Committee Member

David Thomas

Fourth Committee Member

Dongxiao Zhang

Abstract

In the past decades, a series of small peptides, Protein Transduction Domain (PTD), were discovered to be able to facilitate the delivery of small proteins into living cells. With the specific feature, researchers have successfully delivered some functional proteins into living cells. To fully explore and understand the functions and structures of intracellular proteins, more powerful tools are under demand. Recently, an increasing number of rabbit monoclonal antibodies (RabMAbs) have been approved to able to recognize subtle distinctions between the changes of intracellular proteins status. They could be good tools for researchers with the ability to traverse through cell membrane into living cells.

In this dissertation, a novel delivery technology for RabMAbs was established. Transcriptional activator of transcription (TAT) peptide was utilized as a delivery carrier for RabMAbs. It was demonstrated that RabMAbs could be delivered into living cells by conjugating with TAT peptide. Different cell lines, including adherent and suspension cells, were tested for the delivery of RabMAbs. The delivery process was studied in terms of incubation concentration and time, and an optimal delivery condition was established.

To investigate the biological function of delivered RabMAbs inside cytoplasm, three RabMAbs against actin, procaspase-3 and NF-κB respectively were studied. Their binding activities after delivery were verified via sandwich-ELISA data. The immunofluorescent staining of the delivered RabMAb against actin showed it specifically bound to the actin filament in its native morphology. The quantitative analysis of the delivered RabMAb against procaspase-3 showed that approximately 60% of delivered antibody bound to the antigen proteins. The delivered RabMAb against NF-KB apparently blocked the nuclear translocation of NF-KB introduced by TNF-a. The success of delivering the three rabbit monoclonal antibodies with binding or inhibiting functions demonstrated the feasibility of delivering various RabMAbs into living cells by TAT peptide for studying the biological functions of intracellular proteins.

Furthermore, to overcome the efficiency and cost issues of the RabMAb delivery system, a universal delivery platform for RabMAbs was developed. This platform uses goat-anti-rabbit polyclonal antibody conjugated with TAT peptide as delivery vehicle. It was confirmed that the goat-anti-rabbit polyclonal antibody modified with TAT peptide was able to capture RabMAbs and deliver RabMAbs into living cells by the conjugated TAT peptide. The results provide a promising delivery platform for all RabMAbs.

Pages

156

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