|Miller, Marcia - CITY OF HOPE MEDICAL CTR|
|Goto, Ronald - CITY OF HOPE MEDICAL CTR|
Submitted to: Avian Immunology Group Meeting
Publication Type: Proceedings
Publication Acceptance Date: October 8, 2000
Publication Date: N/A
Technical Abstract: Rfp-Y is a second polymorphic system of Mhc-like genes in the chicken. To express cloned Rfp-Y class I cDNA on the surface of chicken cells two Rfp-Y class I cDNA clones (YFVw*7.1, the YFV allele carried by Line C chickens, and c36f, the YFV allele from UCD Line 330) were FLAG epitope-tagged, sub- cloned into the RCASBP (A) vector in both coding and non-coding orientations, and transfected into DF1 cells. DF1 culture supernatants wer used in turn to infect RP9 cells. Both clones in coding orientation were found by flow cytometry to express substantial quantities of FLAG-tagged protein on intact RP9 cells. Hence both clones encode molecules capable of passing through the ER and arriving at the cell surface. Immunoblots provide evidence for association of the YFV protein with b2-microglobulin. To determine whether allogeneic immune responses can be generated to Rfp-Y class I molecules, we chose to immunize 1515 x 72 chickens with RP9 cells expressing FLAG-tagged YFVw*7.1 (RP9 cells and 1515 x 72 chickens share B and Rfp-Y genotypes and have YFV alleles different from YFVw*7.1). The resulting antisera were found to react specifically with YFVw*7.1- expressing RP9 cells indicating that YFV protein is immunogenic in chickens. We found that the antisera react not only specifically with RP9 cells expressing YFVw*7.1 but also with erythrocytes and white blood cells from line C chickens. Specificity was also shown with N and P line chickens.