An exploration of the chicken class I major histocompatability complex and its relationship to Marek's disease

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 (BF 19 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 BF2/2 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 BF 19 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 BF 21 class I MHC alleles, a new class I MHC sequence designated BF 21P was discovered. Comparison of BF21P with known MHC class I allele sequences indicates BF21P 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 BF21P locus is located on chromosome 16 closely linked to the BF21 allele. Furthermore, the BF21P sequence is not found in the pheasant, northern bobwhite, or masked bobwhite species.