Date of Completion

5-10-2013

Embargo Period

5-10-2013

Advisors

Dr. Paulo Verardi and Dr. Michael Lynes

Field of Study

Animal Science

Degree

Master of Science

Open Access

Open Access

Abstract

Tobacco products, both combustible and smokeless, contain abundant amounts of toxic chemicals and carcinogens capable of causing extensive damage to the body. Tobacco specific nitrosamines, including 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) and N’-nitrosonornicotine (NNN), are among the most potent of these carcinogens and are capable of eliciting cellular transformations within the body after bioactivation by cytochrome P450 enzymes. In order to reduce the harm associated with smokeless tobacco, a recombinant anti-NNK antibody has been constructed from a hybridoma secreting a mouse monoclonal antibody specific for NNK.

In work performed previously, hybridomas were created by constructing a structurally-related benzoyl derivative to facilitate coupling to NNK-carrier proteins, which were then used to immunize BALB/C mice. Splenocytes from mice bearing NNK-specific antibodies were used for hybridoma production and the final product was isolated, characterized and found to secrete a high-affinity anti-NNK monoclonal antibody. In the current study, the heavy and light chain antibody F(ab) fragments were cloned, sequenced and inserted in tandem into an expression vector with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab) with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers.

Major Advisor

Dr. Lawrence Silbart

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