Senior Honors Projects, 2010-current

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Date of Award

Spring 2014

Document Type

Thesis

Degree Name

Bachelor of Science (BS)

Department

Department of Integrated Science and Technology

Advisor(s)

Robert L. McKown

Ronald Raab

Kyle Seifert

Abstract

Purpose: Lacritin is a human tear protein first characterized as a novel secretion enhancing factor from the human lacrimal gland (Sanghi, et. al., 2001). Preclinical animal studies have shown that recombinant human lacritin promotes basal tearing in rabbit eyes upon topical application (Samudre et. al., 2011). Antibodies were produced specific for human lacritin, and a clinical assay was developed to detect and quantify lacritin in human tear samples (Seifert K, et. al., 2012). Lacritin is currently being developed as a new topical therapeutic for the treatment of dry eye; however, a dry eye animal model system is needed to test lacritin prior to human clinical trials. The canine dry eye model is promising in that it is a common naturally occurring disorder with clear clinical parallels to dry eye disease in humans (Kymionis, et. al., 2008). The first objective of the study was to determine if the assay to detect lacritin in human tears could detect a lacritin like protein in canine tears. Then an effort was made to clone, sequence, and purify the canine lacritin protein. The purified canine lacritin will be used for a preclinical study in a healthy canine population to test for stimulation of tear production upon topical application. Methods: Fresh canine lacrimal glands were collected at VA-MD Regional College of Veterinary Medicine. RT-PCR was used to amplify the canine lacritin gene from lacrimal tissue. The gene was cloned into an intein expression vector and sequenced. Canine lacritin was expressed in E. coli, purified by chitin and DEAE chromatography, and analyzed by SDS PAGE. Results: A canine lacritin gene homolog was amplified from lacrimal tissue and sequenced. The canine lacritin gene was expressed in E. coli and purified to a single protein band on SDS PAGE. Conclusions: The gene for canine lacritin has been successfully cloned and sequenced. Protein sequence analysis reveals the canine lacritn is 29% identical to human lacritin. The canine lacritin DNA has been expressed in E coli, and purified to greater than 95% purity.

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