Ispitivanje alergenih osobina rekombinantne glukanaze banane (Musa acuminata)

Abstract

Аlergije na hranu se definišu kao nepoželjne imunološke reakcije koje se javljaju svaki put prilikom konzumiranja odrеđene hranljive namirnice. Danas alergije na hranu predstavljaju značajan zdravstveni problem, i smatra se da oko 5% dece i 3-4% odraslih ljudi pati od ovog poremećaja, a uočena je i tendencija rasta broja alergičnih pacijenata. Istovremeno, čak i samo dijagnostifikovanje alergije na hranu predstavlja izazov, s obzirom da se u tu svrhu trenutno upotrebljavaju alergeni ekstrakti, koji su uzrok slabe specifičnosti i osetljivosti dijagnostičkih testova. Rekombinantna DNK tehnologija je razvijena tokom 80-tih godina prošlog veka i pružila je mogućnost proizvodnje rekombinantnih proteina koji bi bili ekvivalentni prirodnim alergenima. Ovi proteini se proizvode kao biohemijski definisani molekuli sa ujednačenim strukturnim i imunološkim osobinama. Do danas je nekoliko stotina različitih alergena klonirano i eksprimirano u obliku rekombinantnih proteina. Ovi rekombinantni alergeni su omogućili znatno detaljniju dijagnostiku senzitizacionih profila alergičnih pacijenata. Iz ovog razloga je proizvodnja dobro okarakterisanih rekombinantnih proteina postala važno pitanje u farmaceutskoj industriji. Banana (Musa acuminata) je veoma popularno voće širom sveta i prisutno je tokom cele godine. Međutim, 1991. godine su prvi put opisani simptomi alergijske reakcije na ovo voće. Do danas je identifikovano 5 proteina koji čine molekulsku osnovu alergije na bananу. Ovi proteini su označeni kao Mus a 1 do 5. Alergen Mus a 5 predstavlja β-1, 3-glukanazu iz PR-2 porodice proteina i učestvuje kako u odbrani biljke, tako i u mnogim drugim raznolikim fiziološkim i razvojnim procesima. Tokom izrade ove disertacije, β-1, 3-glukanaza je proizvedena kao heterologi protein u bakteriji Escherichia coli. Gen glukanaze iz banane (GenBank GQ268963) je ukloniran u ekspresioni vektor pGEX-4T sa glutation-S-transferazom (GST). BL21 ćelije transformisane sa GST-Mus a 5 konstruktom su korišćene za proizvodnju proteina. Uslovi za ekspresiju proteina su optimizovani variranjem temperature (25, 30 i 37°C) i dužine trajanja proteinske sinteze (3, 6 i 12 h)...

Food allergy is defined as an adverse health effect arising from a specific immune response that occurs reproducibly on exposure to a given food. Currently, food allergy is a major health problem, with an estimated prevalence of about 5% in young children and 3-4% in adults, and the prevalence is increasing. Food allergies present a challenge in the field of diagnostics, since the currently employed allergen extracts frequently give rise to poor specificity and sensitivity of tests. Recombinant DNA technology developed in the 1980s has provided the means for producing allergens that are equivalent to their natural counterparts. The proteins are produced as biochemically defined molecules with consistent structural and immunologic properties. Several hundred allergens have been cloned and expressed as recombinant proteins, and they can provide the means for making a very detailed diagnosis of a patient's sensitization profile. The production of recombinant proteins in a well-characterized form has become an important issue in the pharmaceutical industry. Banana (Musa acuminata) is a very popular fruit worldwide. It is available year round and is very present in human nutrition. However, in 1991 symptoms of allergic reaction to banana were described for the first time. The molecular basis of allergic reactions to banana has been ascribed to five proteins, designated Mus a 1 to 5. The Mus a 5 allergen is a β-1, 3-glucanase belonging to the PR-2 family of proteins and is involved not only in plant defense, but also in diverse physiological and developmental processes. In this thesis banana β-1,3-glucanase was produced as a heterologous protein in Escherichia coli. Its gene (GenBank GQ268963) was cloned into a pGEX-4T expression vector as a fusion protein with glutathione-S-transferase (GST). BL21 cells transformed with the GST-Mus a 5 construct were employed for production of the protein. The conditions for protein expression were optimized by varying the temperature (25, 30 and 37 °C) and duration of protein expression (3, 6 and 12 h)...