Open AccessPRODUCTION AND CHARACTERIZATION OF RECOMBINANT PROTEINS CONTAINING BET V 2 ANTIGENIC EPITOPES OF BETULA PENDULA
Author(s) -
А. В. Чернышева,
V V Tyutyaeva,
Anastasia V. Pivovarova,
I. M. Andreev,
М Н Санков,
Yulia Kuzmenko,
Elizaveta Starodubova,
А. И. Мартынов,
В.Л. Карпов
Publication year - 2012
Publication title -
rossijskij allergologičeskij žurnal
Language(s) - English
Resource type - Journals
eISSN - 2686-682X
pISSN - 1810-8830
DOI - 10.36691/rja690
Subject(s) - recombinant dna , microbiology and biotechnology , affinity chromatography , flag tag , myc tag , allergen , biology , escherichia coli , complementary dna , expression vector , inclusion bodies , gene , chemistry , biochemistry , fusion protein , allergy , immunology , enzyme
Aim of investigation. Production of immunologically active recombinant protein of Bet v 2 allergen ofbirch pollen. Materials and methods. mRNA was isolated from a sample ofbirch pollen. cDNA library was derived using SMART technology. Gene Bet v 2 was amplified by means of PCR with primers from the cDNA. The resulting PCR fragment of the gene was cloned into the vector pET29b(+). The recombinant protein Bet v 2 was expressed in cells E. coli, transformed with a plasmid. The recombinant protein was purified using NiNTA agarose. Immunological activity of the recombinant protein Bet v 2 was measured by ELISA and immunoblot methods. Results. The production system of the recombinant allergen Bet v 2 preparation suitable for immunological tests was developed during the research project. In the first phase allergen Bet v 2 gene was cloned from birch pollen collected in Russia. The gene was inserted into the vector pET29(+) for expression in bacterial cells. The expression cell strain E. coli was obtained with this plasmid. The synthesis of the recombinant protein that accumulates in inclusion bodies was activated in bacterial cells. The procedure of recombinant protein Bet v 2 isolation from inclusion bodies was developed by one round of chromatography purification. The recombinant protein isolation was carried out by chromatography on Niagarose. The highly purified preparation of the recombinant allergen was obtained as a result. The recognition of the recombinant protein Bet v 2 by sera varied in ELISA, indicating a different degree of patients sensitization to this allergen. In the immunoblot test the preparation was active only in 15% of cases. Apparently, reactive epitopes of the allergen are mainly conformational ones and are active in ELISA test, whereas linear epitopes, that are active in immunoblot, are in the minority. Conclusion.The system for production of recombinant allergen Bet v 2 preparation suitable for immunological tests has been developed.