Estabilishment of SSCP profile reference panel for analysis of the major histocompatibility complex class II DRB gene in the red deer (Cervus elaphus)

Abstract

Imunološko prepoznavanje vlastitog od stranog djelomično je kontrolirano setom gena unutar glavnog sustava tkivne podudarnosti (MHC). Ti geni kodiraju sustav membranskih glikoproteinskih receptora čija je glavna uloga prepoznati strane proteine, prezentirati ih specijaliziranim imunosnim stanicama, te pokrenuti imunološki odgovor. Veća varijabilnost genskih lokusa MHC omogućuje prepoznavanje šireg spektra antigena, pa posljedično i bolju obranu od patogena. Istraživanja varijabilnosti lokusa MHC analizom sekvenci fragmenata DNA umnoženih PCR reakcijom, obično uključuju i molekularno kloniranje. Uvođenjem metode analize polimorfizma konformacije jednolančane DNA (SSCP) postupak utvrđivanja genske varijabilnosti bio bi jednostavniji, brži i jeftiniji smanjujući potrebu za molekularnim kloniranjem i dodatnim sekvenciranjem. U ovom istraživanju, po prvi puta u Hrvatskoj, uvela sam metodu SSCP u istraživanje raznolikosti alela lokusa DRB, te uspostavila referentni panel SSCP profila spomenutih alela u jelena (Cervus elaphus). U ispitivanim uzorcima utvrdila sam postojanje ukupno sedam različitih alela, te postojanje dupliciranog lokusa DRB. Na referentnom panelu, svaki od pojedinačnih alela pokazuje jedinstven SSCP profil, ali utvrđeno je i preklapanje pojedinih vrpci različitih alela. Kod nekoliko odabranih uzoraka, analiza SSCP izravno umnoženih fragmenata alela potvrdila je rezultate dobivene molekularnim kloniranjem i sekvenciranjem. No, za rutinsku uporabu SSCP panela u procjeni imunogenetičke varijabilnosti jelena u Hrvatskoj, trebalo bi metodu dodatno optimizirati i unaprijediti.Immunological self/nonself recognition is partly controlled by a set of genes in the major histocompatbility complex (MHC). These genes encode complex of membrane glicoproteine receptors which primary role is to recognize foreign proteins, present them to specialist immune cells and initiate an immune response. Increased variability of MHC gene loci enables identification of a broader range of antigens, and consequently a better defense against pathogens. Investigation of MHC variability by analyses of sequence fragments multiplied by PCR reaction, usually includes molecular cloning. By introducing SSCP (single strand conformation polymorphism) method, the process of determining gene variability would be simpler, faster and cheaper and reduce the need for molecular cloning and additional sequencing step. In this experment, for the first time in Croatia, I introduced the SSCP method to explore the diversity of alleles of the DRB locus and estabilished a reference panel of the SSCP profile of the mentioned alleles in the red deer (Cervus elaphus). In analyzed samples, I determined the presence of seven unique alleles in total and a duplication of DRB locus. On the reference panel, each of the alleles showed a unique SSCP profile; however, the overlapping of some indivudual bands of different alleles was found. In chosen representative samples, SSCP analysis of directly amplified allele fragments confirmed the results obtained by molecular cloning and sequencing. Nonetheless, the use of SSCP panel in routine determination of immunigenetic variability of deer in Croatia should be further optimized and improved