Conway, Thomas P.
Ph.D. (Doctor of Philosophy)
Department of Biological Sciences
Major histocompatability complex; Chickens--Genetics; Chickens--Diseases--Illinois; Genetic recombination
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.
O'Sullivan, Sean Kevin, "An exploration of the chicken class I major histocompatability complex and its relationship to Marek's disease" (2003). Graduate Research Theses & Dissertations. 1303.
xi, 100 pages (some color pages)
Northern Illinois University
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