Publication Date

2003

Document Type

Dissertation/Thesis

First Advisor

Conway, Thomas P.

Degree Name

Ph.D. (Doctor of Philosophy)

Department

Department of Biological Sciences

LCSH

Major histocompatability complex||Chickens--Genetics||Chickens--Diseases--Illinois||Genetic recombination

Abstract

The β₂M and the BF¹⁹ and BF²¹ class I MHC genes of the chicken have been cloned and sequenced. These genes were inserted into pIZ/V5-His (β₂M) and pIB/V5-His (BF¹⁹ or BF²¹) expression vectors and High Five insect cells were transfected producing two recombinant cell lines: H5-BF19 and H5-BF21. The BF¹⁹ and BF²¹ class I MHC alleles were expressed on these insect cell lines. H5-BF19 produced anti-BF¹⁹ and H5-BF21 produced anti-BF²¹ antibodies when injected into a BF^(2/2) chicken. Furthermore, using immunofluorescence analysis, conventional anti-BF¹⁹ and anti-BF²¹ antisera specifically stained the H5-BF19 and H5-BF21 cell lines. Due to the absence of the endoplasmic reticulum transport found in vertebrate cells, the H5-BF19 and H5-BF21 insect cell lines do not insert peptide epitopes into class I MHC glycoproteins that are expressed on the surface of insect cells. Incubation of H5-BF19 cells with nine-amino-acid peptides, derived from the sequence of the glycoprotein B gene of Marek's disease virus, stabilized the BF¹⁹ class I glycoprotein and increased surface expression on the H5-BF19 cell line as measured by immunofluorescence. The H5-BF19 insect cells were loaded with three different glycoprotein B epitopes and were incubated with cytotoxic T lymphocytes produced to glycoprotein B. H5-BF19 cells, loaded with peptide 1 (GRRVAAKML), were specifically killed by anti-glycoprotein B CTL. During the cloning and sequencing of the BF¹⁹ and BF²¹ class I MHC alleles, a new class I MHC sequence designated BF^(21P) was discovered. Comparison of BF^(21P) with known MHC class I allele sequences indicates BF^(21P) has 80 to 85% sequence homology with BF¹⁹ and BF²¹. BF^(21P) cDNA contains a number of insertions that produce three internal stop codons and is designated a pseudogene. PCR primers specific for BF^(21P) were designed and used for genetic analysis of the Briles chicken lines maintained at Northern Illinois University, DeKalb, Illinois. The data indicate that the BF^(21P) locus is located on chromosome 16 closely linked to the BF²¹ allele. Furthermore, the BF^(21P) sequence is not found in the pheasant, northern bobwhite, or masked bobwhite species.

Comments

Includes bibliographical references (pages [94]-100)

Extent

xi, 100 pages (some color pages)

Language

eng

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

Media Type

Text

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