RESUMO
In order to obtain nucleotides aptamers bind to IgE, 80 bp nucleotides single-stranded DNA library containing 40 random nucleotides was designed and synthesized. Oligonucleotides that bind to human Cepsilon3-Cepsilon4 protein were isolated from ssDNA pools by the systematic evolution of ligands by exponential enrichment (SELEX) method using nitrocellulose filters as screening medium. Through the optimization of critical PCR and asymmetric PCR parameters including annealing temperature, cycles, and molar ratios of target protein and ssDNA etc, a suitable screening system was established. The aptamers of Cepsilon3-Cepsilon4 protein with high affinity and high specificity were identified by ELISA with biotin-streptavidin-horseradish peroxidase system, and its primary sequence and second structure were analyzed by DNAMAN package and DNA folding sever after being cloned and sequenced. Moreover, target protein was bound to one aptamer and another aptamer modified with biotion together forming a sandwich-like complex, which was captured in microwell to detect IgE concentration using the optimal combination in the sandwich method named enzyme-linked aptamers sorption assay (ELASA). The method could be used for the quantitative detection of human IgE, and whose sensitivity reached to 120 ng x mL(-1).
Assuntos
Humanos , Aptâmeros de Nucleotídeos , Química , Genética , Sequência de Bases , DNA de Cadeia Simples , Química , Cadeias épsilon de Imunoglobulina , Química , Genética , Oligonucleotídeos , Química , Técnica de Seleção de Aptâmeros , Métodos , Sensibilidade e EspecificidadeRESUMO
Allergic diseases have become global social health problems. The binding of IgE with its high affinity receptor FcepsilonRI plays a key step in I-type allergy. Recently, more and more key molecules on the IgE/FcepsilonRI signaling transduction pathway were to be the drug candidates against allergic diseases, with in-depth study of FcepsilonRI signal pathway gradually. The main drugs include molecule antibodies, peptides, vaccines, fusion proteins, small molecules, and other drugs related to IgE/FcepsilonRI. The recent progress in the study of mechanisms of representative drugs targeting on IgE/FcepsilonRI signaling pathway was reviewed in this article.